Erythrocyte-derived extracellular vesicles and proteins associated with such vesicles as biomarkers for parkinson&#39;s disease

ABSTRACT

The present description relates to methods for clinically assessing Parkinson&#39;s disease in a subject using protein biomarkers of erythrocyte-derived extracellular vesicles (EEV).

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.16/484,073, which is the U.S. National Stage of InternationalApplication No. PCT/CA2018/050150, filed Feb. 9, 2018, which designatesthe U.S., published in English, and claims the benefit of U.S.Provisional Application No. 62/457,350, filed Feb. 10, 2017. The entireteachings of the above applications are incorporated herein byreference.

INCORPORATION BY REFERENCE OF MATERIAL IN XML

This application incorporates by reference the Sequence Listingcontained in the following eXtensible Markup Language (XML) file beingsubmitted concurrently herewith:

-   -   a) File name: 54931003003_Sequence_Listing.xml; created Apr. 25,        2023, 15,256 Bytes in size. The computer readable form is        incorporated herein by reference.

SEQ Uniprot ID NO: Protein sequence of: ID 1 Axin interactor,dorsalization-associated protein Q96BJ3 2 Alpha/beta hydrolasedomain-containing protein Q96IU4 14B 3 Glutamine-dependent NAD(+)synthetase E9PNF5 4 Dihydropteridine reductase P09417 5 Alcoholdehydrogenase [NADP(+)] P14550 6 CB1 cannabinoid receptor-interactingprotein 1 B8ZZB8 7 Ubiguitin carboxyl-terminal hydrolase 24 Q9UPU5 8 ATPsynthase subunit alpha, mitochondrial K7EQH4

BACKGROUND

Parkinson's disease (PD) is one of the most common neurodegenerativedisorders affecting millions of people worldwide. Definite diagnosis forPD can only be made postmortem, for instance, by the characteristicaccumulation of the protein alpha-synuclein into Lewy body inclusionsobserved within neurons. Currently, the diagnosis of PD is based onfitting observed symptoms and their severity into clinical rating scalessuch as the Unified Parkinson's Disease Rating Scale (UPDRS) or theHoehn & Yahr scale. Current clinical assessments are subjective,however, and would benefit from improved methods of clinically assessingPD, particularly at early stages of the disease when therapeutic optionsare likely to be most efficient.

SUMMARY

The present description relates to Parkinson's disease. Moreparticularly, the present description relates to extracellular vesiclesoriginating from erythrocytes, and protein biomarkers associatederythrocyte-derived extracellular vesicles, for use in clinicallyassessing Parkinson's disease in a subject.

The present description relates to the proteomic analysis oferythrocyte-derived extracellular vesicles (EEV) from healthy controlsubjects, subjects with mild Parkinson's disease, and subjects withmoderate Parkinson's disease, as well as the identification of proteinbiomarkers for which expression within EEV was found to be significantlymodified therebetween. Accordingly, the present description generallyrelates to methods of clinically assessing Parkinson's disease based onthe detection and/or quantification of the expression level of one ormore protein biomarker(s) within EEV.

In some aspects, the present description may relate to one or more ofthe following items:

-   -   1. An in vitro method for clinically assessing Parkinson's        disease, the method comprising obtaining a preparation of        isolated erythrocyte-derived extracellular vesicles (EEV) from a        blood sample of a subject having or suspected of having        Parkinson's disease; and determining the expression level(s) of        one or more protein biomarker(s) in the preparation of isolated        EEV, wherein the one or more protein biomarker(s) comprises: (i)        Alpha/beta hydrolase domain-containing protein 14B, if the        subject has or is suspected of having mild or moderate        Parkinson's disease; (ii) Alcohol dehydrogenase [NADP(+)], if        the subject has or is suspected of having mild Parkinson's        disease; (iii) ATP synthase subunit alpha, mitochondrial, if the        subject has or is suspected of having moderate Parkinson's        disease; or (iv) any combination of (i) to (iii), wherein the        expression level of one or more of the protein biomarker(s) is        indicative of, or correlates with, the subject's Parkinson's        disease state.    -   2. An in vitro method for clinically assessing Parkinson's        disease, the method comprising: obtaining a preparation of        isolated erythrocyte-derived extracellular vesicles (EEV) from a        blood sample of a subject having or suspected of having        Parkinson's disease; and determining the expression level(s) of        one or more protein biomarker(s) in the preparation of isolated        EEV, wherein the expression level of one or more of the protein        biomarker(s) is indicative of, or correlates with, the subject's        Parkinson's disease state.    -   3. The method of item 2, wherein the one or more protein        biomarker(s) comprises at least one of: (a) Axin interactor,        dorsalization-associated protein; (b) Alpha/beta hydrolase        domain-containing protein 14B; (c) Glutamine-dependent NAD(+)        synthetase; (d) Dihydropteridine reductase; (e) Alcohol        dehydrogenase [NADP(+)]; (f) CB1 cannabinoid        receptor-interacting protein 1; (g) Ubiquitin carboxyl-terminal        hydrolase 24; and (h) ATP synthase subunit alpha, mitochondrial.    -   4. The method of item 3, wherein the one or more protein        biomarkers comprises at least two of (a) to (h).    -   5. The method of item 3, wherein the one or more protein        biomarkers comprises at least three of (a) to (h).    -   6. The method of item 3, wherein the one or more protein        biomarkers comprises at least four of (a) to (h).    -   7. The method of item 3, wherein the one or more protein        biomarkers comprises at least five of (a) to (h).    -   8. The method of item 3, wherein the one or more protein        biomarkers comprises at least six of (a) to (h).    -   9. The method of item 3, wherein the one or more protein        biomarkers comprises at least seven of (a) to (h).    -   10. The method of any one of items 2 to 9, wherein the one or        more protein biomarkers comprises (a).    -   11. The method of any one of items 2 to 10, wherein the one or        more protein biomarkers comprises (b).    -   12. The method of any one of items 2 to 11, wherein the one or        more protein biomarkers comprises (c).    -   13. The method of any one of items 2 to 12, wherein the one or        more protein biomarkers comprises (d).    -   14. The method of any one of items 2 to 13, wherein the one or        more protein biomarkers comprises (e).    -   15. The method of any one of items 2 to 14, wherein the one or        more protein biomarkers comprises (f).    -   16. The method of any one of items 2 to 15, wherein the one or        more protein biomarkers comprises (g).    -   17. The method of any one of items 2 to 16, wherein the one or        more protein biomarkers comprises (h).    -   18. The method of any one of items 2 to 17, further comprising        determining the expression level(s) of one or more of the EEV        protein(s) listed in Table 4, wherein the one or more EEV        protein(s) does not comprise (a) to (h).    -   19. The method of any one of items 2 to 18, further comprising        normalizing the expression level(s) of the one or more protein        biomarker(s) to one or more of the EEV protein(s) as defined in        item 18.    -   20. The method of any one of items 1 to 19, wherein the        preparation of isolated EEV is obtained after inducing the        calcium-dependent production of EEV from activated erythrocytes        in the blood sample of the subject.    -   21. The method of any one of items 1 to 20, wherein the        preparation of isolated erythrocyte-derived extracellular        vesicles (EEV) is obtained by separating the EEV by flow        cytometry, differential centrifugation, nanomembrane        ultrafiltration, immunoabsorbent capture, size-exclusion        chromatography, ultracentrifugation, magnetic activated cell        sorting (MACS), nanoparticle tracking analysis, light        scattering, electrophoretic light scattering, dynamic light        scattering, electron microscopy, or any combination thereof.    -   22. The method of any one of items 1 to 21, wherein the EEV are        CD235a+ extracellular vesicles.    -   23. The method of any one of items 1 to 22, wherein the EEV are        TSG101+, Rabs+, CD9+, CD63+, CD81+, or any combination thereof.    -   24. The method of any one of items 1 to 23, wherein the EEV are        between about 20 nm and about 1000 nm in diameter.    -   25. The method of any one of items 1 to 24, wherein said EEV are        greater than about 100 nm in diameter.    -   26. The method of any one of items 1 to 25, further comprising        removing hemoglobin from the preparation of isolated EEV prior        to determining the expression level(s) of the one or more        protein biomarker(s).    -   27. The method of any one of items 1 to 26, wherein determining        the expression level(s) of one or more of the protein        biomarker(s) in the preparation of isolated EEV comprises        contacting the protein biomarker with an antibody directed        against the protein biomarker.    -   28. The method of any one of items 1 to 27, wherein determining        the expression level(s) of one or more of the protein        biomarker(s) in the preparation of isolated EEV comprises mass        spectrometry.    -   29. The method of item 28, wherein determining the expression        level(s) of one or more of the protein biomarker(s) in the        preparation of isolated EEV comprises nano liquid chromatography        tandem mass spectrometry (nanoLC-MS/MS).    -   30. The method of any one of items 1 to 29, wherein clinically        assessing Parkinson's disease comprises diagnosing Parkinson's        disease.    -   31. The method of any one of items 1 to 30, wherein clinically        assessing Parkinson's disease in the subject comprises staging        Parkinson's disease.    -   32. The method of any one of items 1 to 31, wherein clinically        assessing Parkinson's disease comprises monitoring the        progression of Parkinson's disease.    -   33. The method of any one of items 1 to 32, wherein clinically        assessing Parkinson's disease comprises monitoring the        effectiveness of treatment of a Parkinson's disease subject.    -   34. The method of item 32 or 33, comprising determining the        expression level(s) of one or more of the protein biomarker(s)        in a preparation of isolated EEV from a further blood sample of        the subject obtained at a later point of time.    -   35. A method for analyzing proteins of erythrocyte-derived        extracellular vesicles (EEV), the method comprising: (i)        isolating erythrocyte-derived extracellular vesicles (EEV) from        a blood sample of a subject and forming an EEV preparation        therefrom; (ii) extracting proteins from the EEV        preparation; (iii) removing hemoglobin from the EEV preparation        to obtain a hemoglobin-free EEV preparation; and (iv) detecting        EEV proteins present in the hemoglobin-free EEV preparation.    -   36. The method of item 35, wherein: step (iii) comprises        removing a hemoglobin-containing fraction of proteins from the        EEV preparation to obtain a hemoglobin-depleted EEV preparation        and a hemoglobin-rich fraction; and step (iv) comprises        detecting EEV proteins present in the hemoglobin-free EEV        preparation and EEV proteins present in the hemoglobin-rich        fraction.    -   37. The method of item 35 or 36, wherein the EEV proteins        detected comprise the protein biomarkers as defined in any one        of items 3 to 17.    -   38. The method of any one of items 35 to 37, wherein the        preparation of isolated EEV is obtained after inducing the        calcium-dependent production of EEV from activated erythrocytes        in the blood sample of the subject.    -   39. The method of any one of items 35 to 38, wherein the        preparation of isolated erythrocyte-derived extracellular        vesicles (EEV) is obtained as defined in item 21.    -   40. The method of any one of items 35 to 39, wherein the EEV are        as defined in any one of items 21 to 24.    -   41. The method of any one of items 35 to 40, wherein the EEV        proteins are detected as defined in any one of items 26 to 28.    -   42. Use of one or more erythrocyte-derived extracellular vesicle        (EEV) protein biomarker(s) as defined in any one of items 3 to        17 for clinically assessing Parkinson's disease in a subject.    -   43. The use of item 42, wherein the clinical assessment is as        defined in any one of items 30 to 33.    -   44. A method of treating a subject with Parkinson's disease, the        method comprising: (i) clinically assessing the subject        according to the method of any one of items 1 to 41; and (ii)        beginning or modifying the subject's Parkinson's disease        treatment based on the clinical assessment in (i).

General Definitions

Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., arepresented merely for ease of reading the specification and claims. Theuse of headings or other identifiers in the specification or claims doesnot necessarily require the steps or elements be performed inalphabetical or numerical order or the order in which they arepresented.

The use of the word “a” or “an”, when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one” butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed to determinethe value. In general, the terminology “about” is meant to designate apossible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unlessindicated otherwise, use of the term “about” before a range applies toboth ends of the range.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, un-recitedelements or method steps.

Other objects, advantages and features of the present description willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIGS. 1A-1F. Optimization of EV detection: controls for flow cytometry.FIG. 1A: To properly set the EV gate, fluorescent silica beads of 100 nm(Red), 500 nm (Blue) and 1000 nm (Yellow) were acquired on a flowcytometer Canto II modified with a FSC-PMT small particles option. TheEV gate was used throughout the experiments. FIG. 1B: Serial dilutions(1, 2, 4 and 10) of erythrocyte-derived EV (EEV) to confirm thelinearity of the quantification. FIG. 1C: FSC-PMT/SSC gates ofplatelet-free plasma (PFP) stained with annexin V and respectivefluorochrome-conjugated antibodies directed against erythrocyte(CD235a+), endothelial (CD31+/CD41−)/platelets (CD41+) and leukocytes(CD14+CD45+, monocytes; CD15+CD45+, granulocytes)-derived EV. Controlsfor EV labeling. FIG. 1D: Treatment with the ion chelator EDTA inhibitedthe binding of annexin V to phosphatidylserine. FIG. 1E: Minimalbackground was observed using antibodies in absence of PFP. Thisbackground was subtracted from all subsequent EV quantifications. FIG.1F: EV sensitivity to 0.5% Triton™ was assessed. Abbreviations: AnnV,annexin V; FSC PMT-H, forward scatter photomultiplier; PBS, phosphatebuffered saline; PFP, platelet free plasma; SSC-H, side scatter.

FIGS. 2A and 2B. EEV: A biomarker of PD state. FIG. 2A shows thecorrelations between the number of erythrocyte-derived extracellularvesicles (EEV; expressed as CD235a+ EV/total number of erythrocytes) andthe Unified Parkinson's Disease Rating Scale (UPDRS) of subjects (n=20).Robust correlations between the number of EEV/total number oferythrocytes and UPDRS scores (PD, n=20), displayed a clear splitbetween mild and moderate patients, supporting the potential of EEV as abiomarker for disease state. Additional information on five patients(identified as 1-5) derived from each correlation is provided (number ofEEV/total number of erythrocytes and levodopa dose equivalent) toillustrate that levodopa dosing cannot account for differences in EEVcounts. Note that patient no. 5, which falls outside the confidenceboundary, is the only patient on a regimen of anti-inflammatory drugs tomanage arthritis. FIG. 2B shows the results of a similar analysis as inFIG. 2A, but performed on Huntington's disease subjects (n=42) using theUnified Huntington's Disease Rating Scale (UHDRS). The numbers of EEVare expressed as CD235a+ EV/total number of erythrocytes. Nostatistically significant correlations were found between the number ofEEV/total number of erythrocytes and UHDRS scores. This argues in favorof EEV being a specific biomarker of PD. Distributions were determinedusing unpaired t-test with Welch's correction (PD) or one-way ANOVA(HD). Correlations were determined using Pearson's correlation, *p<0.05.Abbreviations: CD235a, glycophorin A; CTRL, Controls; EEV,erythrocyte-derived extracellular vesicle; EV, extracellular vesicle;HD, Huntington's disease; LEDD, Levodopa equivalent daily dose; PD,Parkinson's disease; Pre-HD, Pre-manifest; UHDRS, Unified Huntington'sDisease Rating Scale; UPDRS, Unified Parkinson's Disease Rating Scale.

FIGS. 3A and 3B. Detection of normal and phosphorylated α-Syn in EEV.FIG. 3A: Representative scanning electron microscopy observations ofresting and activated erythrocytes (treated with calcium ionophoreA23187 to generate EEV) in both PD patients and healthy sex- andage-matched CTRL. Scale bar: 2 μm. FIG. 3B: Representative transmissionelectron microscopy images of immunogold labeling for α-Syn and α-SynpS129 in activated erythrocytes and EEV (some examples delineated bydotted lines). Arrowheads point to positive immunolabeling for eitherα-Syn or α-Syn pS129. Scale bar: 100 nm. FIG. 3C: Quantification ofα-Syn in EEV as detected by transmission electron microscopy andexpressed as the percentage of EEVs positive for α-Syn/total number ofEEV in healthy sex- and age-matched CTRL and PD patients (n=100erythrocytes sampled in n=3 CTRL and n=3 PD). FIG. 3D: Quantification ofα-Syn in EEV by ELISA assay in healthy sex- and age-matched CTRL, mildand moderate stage patients selected according to their H&Y stage (n=4erythrocytes per group; n=13 EEV per group) revealing the absence ofmeasurable changes in α-Syn levels between PD and healthy sex- andage-matched CTRL. Statistical analyses were performed using aMann-Whitney U test (FIG. 3C) or a Kruskal-Wallis ANOVA (FIG. 3D).Abbreviations: α-Syn, α-synuclein; α-Syn pS129, α-synucleinphosphorylated Serine 129; CTRL, Control; EEV, erythrocyte-derivedextracellular vesicle; H&Y, Hoehn and Yahr; PD, Parkinson's disease.

FIGS. 4A-4D. Specific protein signature of EEV in PD patients. FIG. 4A:NanoLC-MS/MS Label-free analysis of EEV in PD patients and healthyage-matched CTRL (PD, n=4; CTRL, n=4) revealed a total of 818 proteins,with 8 of which the expression was significantly modified as a functionof PD states. FIG. 4B: The 8 differentially expressed proteins arereferenced according to the gene to which they are associated, andfurther separated into 3 groups in relation to their expressionvariations in comparison to CTRL (Group I), mild PD (Group II) ormoderate PD (Group III).

FIG. 4C: Normalized expression (intensity) of the proteins associatedwith the Group I genes AIDA, ABHD14B, and NADSYN1; the Group II genesQDPR, AKR1A1, and CNRIP1; and the Group III genes USP24 and ATP5A1. FIG.4D: Heatmap establishing correlations between disease states and theabundance of the variable proteins. Cold (C) and hot (H) colorsrepresent low and high correlation levels, respectively. The AU p valueis indicated for each node. Protein modulation was determined byunpaired t-tests with Welch's correction using the criteria of a p valueunder 0.05 and a minimum of 2-fold change between groups, *p<0.05,**p<0.01. Abbreviations: AU, Approximately Unbiased; CTRL Control; PD,Parkinson's disease.

FIGS. 5A-5C. Confirmation of EEV proteins selectively modified in PDpatients by Volcano plots. The protein ratios (log 2(ratio)) of thethree comparison (FIG. 5A: mild PD/CTRL, FIG. 5B: moderate PD/CTRL, andFIG. 5C: moderate PD/mild PD) were plotted over the correspondingWelch's test p value (−log 10(p-value)). The graphs display a V shape,as expected, and only the proteins falling outside the limits of a pvalue<0.05 and absolute value of z-score>1.96 (identified by blacklines) were considered as variant proteins (arrows). Two variantproteins were excluded given that they were quantified using only onepeptide. Abbreviations: CTRL: Control; PD, Parkinson's disease.

DETAILED DESCRIPTION

The present description relates to the proteomic analysis oferythrocyte-derived extracellular vesicles (EEV) and the identificationof protein biomarkers whose expression levels within EEV correlate withdifferent Parkinson's disease (PD) states.

In one aspect, the present description relates to a method forclinically assessing Parkinson's disease in a human subject, based onthe expression level of one or more protein biomarker(s) that maycorrelate with or be indicative of the subject's Parkinson's diseasestate. As used herein, the expression “clinically assessing” or“clinical assessment” in the context of PD refers to an evaluation of asubject's PD state, which may or may not occur in a clinical setting,and which may or may not be performed by a health care professional. Forexample, clinically assessing may comprise screening and/or diagnosingPD in a subject having or suspected of having PD, staging a subject'sPD, monitoring the progression of PD in a subject, monitoring the effectof PD medication or treatment (e.g., over time), or any combinationthereof. The use of the methods described herein with other methods forclinically assessing PD subjects is also envisaged.

In some aspects, the methods described herein may comprise obtaining apreparation of isolated erythrocyte-derived extracellular vesicles (EEV)from a blood sample of a subject having or suspected of havingParkinson's disease. As used herein, the expression “extracellularvesicles” (EV) refers to subcellular membrane vesicles found in theextracellular environment (e.g., bodily fluids) that originate fromcells, and which range in size from about 20 nm to about 1000 nm. EV maycomprise exosomes, microvesicles (MV), multivesicular endosomes (MVE),or vesicles produced by apoptotic bodies, or any combination thereof, aswell as other types of extracellular vesicles. Whereas the majority ofthe circulating EV that are detected by flow cytofluorometric assays arelikely to be MV, we do not completely exclude the potential contributionof larger exosomes or vesicles produced by apoptotic bodies. In someembodiments, the EV of the present description comprise vesicles betweenabout 30, 40, 50, 60, 70, 80, 90, or 100 nm to about 500, 600, 700, 800,900, or 1000 nm in size. In some embodiments, the EV of the presentdescription comprise vesicles from 100 nm to 1000 nm in size. In someembodiments, the EV of the present description comprise vesicles between150 nm to 1000 nm in size. All EV are composed of membrane proteins andlipids, as well as cytoplasmic components of the cell from which theyoriginate, such as mRNA and miRNA, organelles or infectious particles(e.g., prions, virus). A variety of methods may be used to determine theorigin of EV. For example, cell surface markers (e.g., withimmunolabeling and/or flow cytometry techniques) may be used toidentify, enrich/purify/isolate, and/or quantify EV according to theircell of origin. Examples of such markers include: CD235a+(erythrocytes), CD31+/CD41-(endothelial cells), CD41+ (platelets), CD45+(leukocytes), CD45+ CD14+ (monocytes), and CD45+ CD15+(granulocytes). Ofparticular interest for the present description are markers that arepresent in (or specific for) EEV that may be used to identify,enrich/purify/isolate, and/or quantify EEV from other types of EV.Examples of such EEV markers include endosome or membrane-bondingproteins such as TSG101 and Rabs (enriched in exosomes), tetraspaninssuch as CD9, CD63 and CD81 (enriched in exosomes), golgi andmitochondrial proteins (enriched in MVs and absent in exosomes) (Lotvallet al., 2014). In some embodiments, the EEVs of the present descriptionmay comprise one or more of the EEV protein(s) listed in Table 4.

As used herein, the expression “[marker]+EV” or “[marker]-positive” inrelation to extracellular vesicles refers to the presence ordetectability of that marker in an EV population of interest, regardlessof whether that marker is actually detected (e.g., using animmunolabel). Conversely, the expression “[marker]-EV” or“[marker]-negative EV” refers to the absence or lack of detectability ofthat marker in an EV population of interest, regardless of whether thatmarker is actually detected (e.g., using an immunolabel). For example,the expression “CD235+EV” or “CD235a-positive EV” means EV that comprisethe marker CD235a (Glycophorin A).

As used herein, the term “protein biomarker” refers to a molecularindicator that is a polypeptide or protein that is associated with aparticular pathological or physiological state (e.g., PD disease state).For example, the expression “Parkinson's disease biomarker” or “PDbiomarker” refers to a molecular indicator that is associated with thepresence, stage, and/or progression of PD in a subject. Furthermore, theterm “EEV protein biomarker” refers to a protein biomarker that isexpressed in EEV, whose level of expression is associated with aparticular pathological or physiological state (e.g., PD disease state).

In some embodiments, obtaining a preparation of isolated EEV from ablood sample of a subject (e.g., a subject having or suspected of havingParkinson's disease) may involve identifying,enriching/purifying/isolating, and/or quantifying EEV in a blood samplefrom the subject. In some embodiments, the blood samples may beprocessed to obtain platelet-free plasma (PFP), and the preparation ofisolated EEV may be prepared from PFP. As used herein, the terms“enriched”, “purified”, “isolated” and the like, refer to eitherremoving contaminants from a biological sample and/or increasing theconcentration of an analyte of interest (e.g., EEV) in the sample, to anextent that is not found in nature. In some embodiments, identifying,enriching/purifying/isolating, and/or quantifying EEV may involve flowcytometry, differential centrifugation, nanomembrane ultrafiltration,immunoabsorbent capture, size-exclusion chromatography,ultracentrifugation, magnetic activated cell sorting (MACS),nanoparticle tracking analysis, light scattering, electrophoretic lightscattering, dynamic light scattering, electron microscopy or anycombination thereof, or using other techniques that can separatevesicles based on their size and/or surface protein expression.Quantifying EEVs may also be performed by methods such as nanoparticletracking (NTA), biochemical approaches and semi-quantitative electronmicroscopy approaches. In some embodiments, the methods described hereinmay further comprise quantifying the level of EEV in a blood sample froma subject. The quantification of EEV may be expressed as a relativevalue by normalizing the number of EEV (e.g., in terms of the totalnumber of erythrocytes).

In some embodiments, preparations of isolated EEV described herein maycomprise at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% (e.g., by weightor number) of EEV, and/or less than 30%, 25%, 20%, 15%, 10%, 5%, or 1%(e.g., by weight or number) of non-EEV.

In some embodiments, the methods described herein may comprise obtaininga blood sample from a subject that is less than 5 mL, less than 4 mL,less than 3 mL, less than 2 mL, less than 1 mL, less than 900 μL, lessthan 800 μL, less than 700 μL, less than 600 μL, less than 500 μL, lessthan 400 μL, less than 300 μL, less than 200 μL, less than 100 μL, lessthan 50 μL, less than 40 μL, less than 30 μL, less than 25 μL, less than20 μL, or less than 10 μL.

In some embodiments, the methods described herein may comprise detectingor determining the expression level of an EEV protein biomarker that isdifferentially expressed in controls (e.g., non-PD subjects) and/or indifferent PD states (e.g., mild, moderate, or severe), for example basedon the unified Parkinson's disease rating scale (UPDRS). As used herein,the expression “mild PD patients” are defined as patients characterizedby a UPDRS score of lower than 37; “moderate PD patients” are defined aspatients characterized by a UPDRS score of between 37 and and “severe PDpatients” are defined as patients characterized by a UPDRS score ofgreater than 75. In some embodiments, the EEV protein biomarkersdescribed herein may be used, for example, to distinguish between mild,moderate and severe PD patients. In some embodiments, the EEV proteinbiomarkers described herein may be used to distinguish between patientscharacterized by UPDRS scores within different ranges from thosementioned above. As used herein, “control subjects” or “controls” referto non-PD subjects (e.g., healthy subjects).

In some embodiments, the methods described herein comprise determiningthe expression level(s) of one or more protein biomarker(s) in apreparation of isolated EEV, wherein the protein biomarker(s) is/are oneor more of the proteins defined in the Table below.

SEQ ID Related Related Corresponding Groups Protein biomarker NO:accession no. UniProt ID gene name I Axin interactor, dorsalization- 1NP_073742.2 Q96BJ3 AIDA (Control) associated protein Alpha/betahydrolase domain- 2 NP_116139.1 Q96IU4 ABHD14B containing protein 14BGlutamine-dependent NAD(+) 3 EAW74792.1 E9PNF5 NADSYN1 synthetase IIDihydropteridine reductase 4 NP_001293069.1 QDPR QDPR (mild PD) Alcoholdehydrogenase [NADP(+)] 5 AAP36383.1 P14550 AKR1A1 CB1 cannabinoidreceptor-interacting 6 NP_056278.1 B8ZZB8 CNRIP1 protein 1NP_001104571.1 III Ubiguitin carboxyl-terminal hydrolase 7 NP_056121.2Q9UPU5 USP24 (moderate 24 PD) ATP synthase subunit alpha, 8 EAX01470.1K7EQH4 ATP5A1 mitochondrial

In some embodiments, the protein biomarkers identified herein asbelonging to Group I may be differentially expressed in the EEV ofhealthy or non-PD control subjects, as compared to those of mild and/ormoderate PD subjects. In some embodiments, the protein biomarkersidentified herein as belonging to Group II may be differentiallyexpressed in the EEV of mild PD subjects, as compared to those ofhealthy or non-PD control subjects and/or moderate PD subjects. In someembodiments, the protein biomarkers identified herein as belonging toGroup Ill may be differentially expressed in the EEV of moderate PDsubjects, as compared to those of healthy or non-PD control subjectsand/or mild PD subjects.

In some embodiments, the methods described herein may comprisedetermining the expression of one or more of the protein biomarkersdescribed herein, based on the PD state which is to be clinicallyassessed. For example, different protein biomarkers or combinations ofprotein biomarkers may be selected based on, for example, thedifferential expression patterns reported herein (e.g., in Table 3.1 andin FIG. 4C). In some embodiments, the methods described herein maycomprise determining the expression of: (i) Alpha/beta hydrolasedomain-containing protein 14B, if the subject has or is suspected ofhaving mild or moderate Parkinson's disease; (ii) Alcohol dehydrogenase[NADP(+)], if the subject has or is suspected of having mild Parkinson'sdisease; (iii) ATP synthase subunit alpha, mitochondrial, if the subjecthas or is suspected of having moderate Parkinson's disease; or (iv) anycombination of (i) to (iii). In some embodiments, the subject isdetermined to have mild or moderate PD when the expression level ofalpha/beta hydrolase domain-containing protein 14B in the preparation ofisolated EEV from a blood sample of the subject is lower than thatcorresponding to a control subject (i.e., not having PD). In someembodiments, the subject is determined to have mild PD when theexpression level of alcohol dehydrogenase [NADP(+)] in the preparationof isolated EEV from a blood sample of the subject is higher than thatcorresponding to a control subject (i.e., not having PD). In someembodiments, the subject is determined to have moderate PD when theexpression level of ATP synthase subunit alpha, mitochondrial in thepreparation of isolated EEV from a blood sample of the subject is higherthan that corresponding to a control subject (i.e., not having PD). Insome embodiments, the terms “higher” or “lower” may refer to adifference in expression from the reference value of at least 1.5-fold,2-fold, 2.5-fold, 3-fold, or 3.5-fold. In some embodiments, methodsdescribed herein may comprise determining the expression level(s) of atleast one, at least two, at least three, at least four, at least five,at least six, at least seven, or all eight of the protein biomarker(s):

-   -   (a) Axin interactor, dorsalization-associated protein        (represented by SEQ ID NO: 1);    -   (b) Alpha/beta hydrolase domain-containing protein 14B        (represented by SEQ ID NO: 2);    -   (c) Glutamine-dependent NAD(+) synthetase (represented by SEQ ID        NO: 3);    -   (d) Dihydropteridine reductase (represented by SEQ ID NO: 4);    -   (e) Alcohol dehydrogenase [NADP(+)] (represented by SEQ ID NO:        5);    -   (f) CB1 cannabinoid receptor-interacting protein 1 (represented        by SEQ ID NO: 6);    -   (g) Ubiquitin carboxyl-terminal hydrolase 24 (represented by SEQ        ID NO: 7); and    -   (h) ATP synthase subunit alpha, mitochondrial (represented by        SEQ ID NO: 8).

In some embodiments, the expression level of one or more of the proteinbiomarker(s) described herein may be determined by detecting and/orquantifying the presence of a polypeptide fragment of any one of thepolypeptides of SEQ ID NOs: 1-8. Such fragments may be comprise orconsist of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 125, 150,175, 200, or more contiguous amino acids of any one of SEQ ID NOs: 1-8.In some embodiments, the polypeptides of any one of SEQ ID NOs: 1-8 maybe present in the EEV (or a preparation of isolated EEV) as a fragmentof SEQ ID NOs: 1-8. Detection of such fragments are considered withinthe scope of the present description. In some embodiments, a proteinbiomarker described herein may comprise one or more fragments of any oneof SEQ ID NOs: 1-8, for example a fragment comprising or consisting of:residues 127-192 of SEQ ID NO: 1; residues 188-200 of SEQ ID NO: 2;residues 112-260 or residues 349-446 of SEQ ID NO: 3; residues 1-213 ofSEQ ID NO: 4; residues 1-325 of SEQ ID NO: 5; residues 1-110 or residues84-110 of SEQ ID NO: 6; residues 2570-2620 of SEQ ID NO: 7; or residues51-161, residues 111-137, or residues 53-152 of SEQ ID NO: 8.

In some embodiments, methods described herein may comprise determiningthe expression level(s) of at least one, at least two, at least three,at least four, at least five, at least six, at least seven, or all eightof the protein biomarker(s) encoded by the human genes AIDA, ABHD148,NADSYN1, QDPR, AKR1A1, CNRIP1, USP24, and ATP5A1.

In some embodiments, methods described herein may comprise determiningthe expression level(s) of at least one, at least two, at least three,at least four, at least five, at least six, at least seven, or all eightof the protein biomarker(s) defined by accession numbers: NP_073742.2,NP_116139.1, EAW74792.1, NP_001293069.1, AAP36383.1, NP_056278.1,NP_001104571.1, NP_056121.2, and EAX01470.1.

In some embodiments, protein biomarker (a) may be at least 70%, 75%,80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQ IDNO: 1. In some embodiments, protein biomarker (b) may be at least 70%,75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of SEQID NO: 2. In some embodiments, protein biomarker (c) may be at least70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence ofSEQ ID NO: 3. In some embodiments, protein biomarker (d) may be at least70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence ofSEQ ID NO: 4. In some embodiments, protein biomarker (e) may be at least70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence ofSEQ ID NO: 5. In some embodiments, protein biomarker (f) may be at least70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence ofSEQ ID NO: 6. In some embodiments, protein biomarker (g) may be at least70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence ofSEQ ID NO: 7. In some embodiments, protein biomarker (f) may be at least70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence ofSEQ ID NO: 8.

In some embodiments, the methods described herein may comprisedetermining the expression levels of an EEV protein signature comprisingany combination of the protein biomarkers (a) to (h). In someembodiments, the methods described herein may comprise determining theexpression levels of at least one of protein biomarkers (a), (b), and(c) [Group I]; at least one of protein biomarkers (d), (e), and (f)[Group II]; and/or at least one of protein biomarkers (g) and (h) [GroupIII].

In some embodiments, the methods described herein may further comprisecomparing the expression level of the protein biomarker(s) so detectedand comparing the expression level to a reference value corresponding tothat of a control group, non-PD group, mild PD group, moderate PF group,or severe PD group.

In some embodiments, the EEV protein biomarkers described herein do notcomprise alpha-synuclein; and/or the methods described herein do notcomprise detecting and/or quantifying alpha-synuclein.

In some embodiments, the expression level(s) of one or more of theprotein biomarker(s) in the preparation of isolated EEV may comprisecontacting the protein biomarker with an antibody directed against theprotein biomarker (e.g., an immunoassay). In some embodiments, theantibody specifically binds to the polypeptide of any one of SEQ ID NOs:1-8. Antibodies against the proteins of SEQ ID NOs: 1-8 are commerciallyavailable, for example from Novus Biologicals (e.g., catalog numbersNBP1-88323, NBP2-26122, H00055191-M01, H00005860-M02, NBP2-02164,NBP1-86800, NB100-40830, and NBP2-38525, respectively). In someembodiments, antibodies described herein may bind to, or be raisedagainst, a fragment of any one of SEQ ID NOs: 1-8, for example afragment comprising or consisting of: residues 127-192 of SEQ ID NO: 1;residues 188-200 of SEQ ID NO: 2; residues 112-260 or residues 349-446of SEQ ID NO: 3; residues 1-213 of SEQ ID NO: 4; residues 1-325 of SEQID NO: 5; residues 84-110 of SEQ ID NO: 6; residues 2570-2620 of SEQ IDNO: 7; or residues 53-152 of SEQ ID NO: 8. In some embodiments,antibodies described herein may bind to an epitope comprising orconsisting of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous residues ofany one of SEQ ID NOs: 1-8.

As used herein, the term “antibody” may encompass any type of antibody,including but not limited to monoclonal antibodies, polyclonalantibodies, “antigen-binding fragments” (or portion), such as Fab, Fab′,F(ab′)2, Fd, Fv, Fc, etc., of intact antibodies that retain the abilityto specifically bind to a given antigen (e.g., an EEV protein describedherein), an isolated complementarity determining region (CDR),bispecific antibodies, heteroconjugate antibodies, mutants thereof,fusion proteins having an antibody, or antigen-binding fragment thereof,(e.g., a domain antibody), single chain (ScFv) and single domainantibodies (e.g., shark and camelid antibodies), maxibodies, minibodies,intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv,humanized antibodies, chimeric antibodies and any other modifiedconfiguration of the immunoglobulin molecule that includes an antigenrecognition site of the required specificity, including glycosylationvariants of antibodies, amino acid sequence variants of antibodies, andcovalently modified antibodies. The antibodies may be murine, rat,human, or any other origin (including chimeric or humanized antibodies).

In some embodiments, the expression level(s) of one or more of theprotein biomarker(s) in the preparation of isolated EEV may comprisemass spectrometry (e.g., nano liquid chromatography tandem massspectrometry (nanoLC MS/MS)).

In some embodiments, the methods described herein may comprise combiningthe expression of one or more of the protein biomarker(s) in thepreparation of isolated EEV with the quantity of EEV to increase thepower of a biomarker described herein.

In some embodiments, the methods described herein may further comprisedetermining the expression level(s) of one or more of the EEV protein(s)listed in Table 4 (Proteins identified in the EEV proteome), wherein theone or more EEV protein(s) does not comprise (a) to (h). The expressionlevel of one or more EEV proteins that are not differentially expressedbetween PD states may be useful for example as normalization parametersin conjunction with the one or more of the protein biomarkers (a) to(h).

In some embodiments, the methods described herein may comprisedetermining the expression level(s) of one or more of the EEV protein(s)defined by UniProt ID: P02549, P11277, P16157, P02730, P55072, P16452,P11171, P04040, Q13228, B4DT77, Q8WUM4, P35612, P68871, Q00610, P69905,B4DVE7, J3QLD9, P08758, P09525, 075955, P11142, P32119, P00491, P27105,P00918, P23634, C9JIF9, P00915, P63261, Q5VU58, P30041, E7EU23, Q00013,E7EV01, J3KPS3, Q08495, P23276, P69892, P60174, P62258, O75326, O75340,E7EV99, Q5VZU9, P53396, C9J0K6, P04406, P07738, B7Z319, Q86X55, P07195,P23526, Q32Q12, B7Z7A9, P11166, P22303, Q9NP58, P40925, P00352, F2Z2V0,K7EMC9, F5H7S3, A6NN80, P30043, H7BXD5, P04083, P62937, P37837, Q06830,O75131, P00390, E7EQB2, P07384, P02042, P30086, P35613, Q9H0U4, P63092,P48506, P06702, Q9UBV8, P17931, P28066, P07451, E7EQ12, P50895, P28074,G3V5Z7, P25786, G3V1D3, P49247, Q5T9B7, P25789, B4E022, J3QS39, HOY7A7,P28070, Q9H4G4, Q9BY43, P48426, P28289, P07911, Q9GZP4, P78417, P25788,O14818, P08107, H0YD13, P61225, P05109, P23528, Q99808, P84077, P31946,C9JIS1, P53990, Q99497, F5H7U0, B7Z7E9, P62834, P04899, P25325, Q9NP79,P00492, Q16531, P22314, P00441, H7BY58, P10768, P09543, P06733, P26038,O75368, K7EQ48, P25787, P49721, F5H8J2, C9J9P4, P09211, B5MDF5, P07900,K7EQ02, Q13630, F5H0T1, P50502, P20618, P62805, P51148, H7C2G2, J3KQ18,H3BPK3, B4DIT7, O43633, B4DQH4, Q9UN37, I3L397, Q9Y5Z4, Q9UKV8, F5H442,H3BLV0, P02008, E7EPV7, P63104, P02724, U3KQE2, Q9NP59, Q5QPM9, C9J8U2,P61981, Q9UQ80, E5RJR5, Q9NRV9, Q9H444, P10599, Q5VSJ9, Q9UK41, Q9Y311,P61026, Q14974, P27797, P18669, P54725, P30613, P63000, P05164, F5GWY2,P48637, F5H5V4, G5E9R5, P46976, P28072, P26447, F5GXQ0, P08754, Q99436,P62942, U3KQK0, J3QKR3, P01116, P13489, Q08722, Q5T123, Q8WYQ7, O75695,P00167, Q9Y4D1, P11021, H7C1D4, P07737, M0R389, A6NJA2, P10644, Q9BS40,G5EA52, P53004, Q04656, H9KV70, O00299, F8WF69, G3V2F7, F8WDS9, P60891,K7ESE8, H0YNE3, P16930, F8VSD4, P07203, P62328, E5RIW3, MOROY2, P15374,P04921, HOYDI1, B4E220, C9JEN3, F5H2R5, Q53TN4, Q9NZD4, Q8NHG7, Q5JYX0,Q71RC9, E9PNW4, P09105, R4GN98, O75531, Q5T6W5, F5H4Q5, J3QK90, H3BV85,Q9NRX4, H3BS66, E7ESC6, P68402, Q9BRF8, P08246, E9PN50, E7EUC7, B8ZZB8,E9PCS3, P59666, O15400, P00338, P61970, E7EMV0, F5GY90, P61020, Q99828,B4DUA0, C9JTY3, P27348, H0YKZ7, P08238, J3KQP6, A6NMU3, P53985, F6USW4,O14964, P20020, P36959, Q9Y376, Q9Y6M5, Q81Z83, Q99459, P06132, J3KNT0,P49189, H3BNT7, P05023, P34932, K7EMV3, Q81U68, E7ENZ3, Q5TZA2, Q9P203,Q7LBR1, U3KQ56, H0YJ11, Q9UDT6, P09960, Q5HY54, E9PJL5, G3V2U7, E9PQN4,Q9BSL1, Q04917, B7ZBP9, Q8NDC0, P68133, P69891, S4R3Y4, I3L3E4, Q16570,Q5VY30, E7END7, Q5VU59, P17066, Q04760, D6RD66, K7EM02, P14209, E9PIR7,K7EMQ9, P15531, H7BZT4, O00560, Q9BVM4, K7EKH5, P49773, H0YBY6, Q9Y624,B1AKQ8, K7EKN6, 13L0K2, A8MXY0, O14773, E9PNW0, Q5TDH0, Q96JM4, F5GWT9,F2Z3J2, J3QL74, E9PJC7, Q9H936, D6RD63, Q6B0K9, Q31611, H7BY04, Q9UL25,H7C3P7, P08311, E9PE37, G3V1N2, P00387, O75339, P14324, K7EKG2, P02549,P11277, P16157, P55072, P111714, P35579, Q8WUM4, P02730, P16452, P04040,A0A087WVQ6, P35612, P16157, Q14254, P20073, O75955, P53396, P06753,P49368, P236344, P11142, P60709, Q5T4S7, P78371, P28289, P50395, P27105,P68871, P02730, Q00013, J3KPS3, P00352, Q86VP6, P49327, Q13228, P00915,P50991, P50990, P32119, P50995, P69905, P00558, H7BXK9, P08758, P09525,P07900, P48643, P07384, P22314, P04406, Q08495, Q99832, P29144, P30041,E9PM69, P40227, P50570, E7EQB2, P31948, E7ESC6, E7EV99, P62258, P30613,AOAOG2JIW1, Q16531, P11021, F5H2F4, P07195, P45974, O43242, C9J0K6,P08133, Q13200, P23276, P34932, A0A0A0MSI0, Q9Y230, Q5XPI4, P68871,P60174, P00491, C9JIF9, H7BYY1, P35998, P17987, P09543, Q99460, Q9Y4E8,Q90009, P26038, P04083, P30043, P11166, P00918, P06733, Q5TDH0, B0QZ18,O75326, P05164, Q9Y265, P29401, I3L0N3, Q4VB86, P11277, P13716, P07738,P48506, Q99816, O14818, P23526, P61225, O00231, P11413, P00338, Q99808,A6NJA2, Q06323, or any combination thereof.

In some embodiments, the methods described herein may comprisedetermining the expression level(s) of one or more of the EEV protein(s)defined by protein accession number: NP_000017.1, NP_000022.3,NP_000028.3, NP_000034.1, NP_000036.2, NP_000043.4, NP_000058.1,NP_000110.2, NP_000128.1, NP_000134.2, NP_000149.3, NP_000166.2,NP_000169.1, NP_000175.1, NP_000185.1, NP_000230.1, NP_000241.1,NP_000260.1, NP_000261.2, NP_000276.2, NP_000280.1, NP_000282.1,NP_000289.1, NP_000311.2, NP_000333.1, NP_000338.3, NP_000356.1,NP_000365.3, NP_000366.1, NP_000382.3, NP_000389.1, NP_000393.4,NP_000410.2, NP_000411.1, NP_000421.1, NP_000445.1, NP_000471.1,NP_000507.1, NP_000508.1, NP_000509.1, NP_000510.1, NP_000549.1,NP_000550.2, NP_000572.2, NP_000623.2, NP_000628.2, NP_000656.1,NP_000678.1, NP_000680.2, NP_000687.3, NP_000691.1, NP_000692.2,NP_000703.2, NP_000704.1, NP_000792.1, NP_000843.1, NP_000886.1,NP_001001323.1, NP_001001396.1, NP_001001521.1, NP_001002021.2,NP_001002857.1, NP_001002858.1, NP_001002860.2, NP_001003938.1,NP_001003945.1, NP_001005360.1, NP_001005361.1, NP_001005362.1,NP_001005386.1, NP_001005753.1, NP_001007068.1, NP_001007069.1,NP_001007070.1, NP_001007071.1, NP_001008390.1, NP_001008800.1,NP_001009185.1, NP_001009186.1, NP_001009570.1, NP_001010935.1,NP_001010942.1, NP_001013275.1, NP_001013454.1, NP_001017963.2,NP_001019397.1, NP_001019398.1, NP_001019399.1, NP_001019820.1,NP_001020029.1, NP_001020276.1, NP_001020560.1, NP_001020561.1,NP_001026897.1, NP_001026997.1, NP_001027017.1, NP_001028196.1,NP_001028690.1, NP_001028691.1, NP_001028692.1, NP_001029197.1,NP_001029249.1, NP_001030611.1, NP_001034221.1, NP_001034271.1,NP_001034288.1, NP_001034455.1, NP_001034456.1, NP_001034679.2,NP_001034680.2, NP_001034708.1, NP_001034891.1, NP_001035517.1,NP_001035784.1, NP_001035810.1, NP_001035941.1, NP_001036816.1,NP_001036817.1, NP_001036818.1, NP_001055.1, NP_001070654.1,NP_001070956.1, NP_001070957.1, NP_001070958.1, NP_001071643.1,NP_001071645.1, NP_001073379.1, NP_001077086.1, NP_001077861.1,NP_001087.2, NP_001091.1, NP_001092.1, NP_001092006.1, NP_001092925.1,NP_001096137.1, NP_001096138.1, NP_001098985.1, NP_001099000.1,NP_001107606.1, NP_001107607.1, NP_001107608.1, NP_001107609.1,NP_001107610.1, NP_001107611.1, NP_001108628.1, NP_001112362.1,NP_001116370.1, NP_001116423.1, NP_001116849.1, NP_001116850.1,NP_001118.3, NP_001120855.1, NP_001121776.1, NP_001121777.1,NP_001121778.1, NP_001122060.3, NP_001122301.1, NP_001122302.1,NP_001122303.1, NP_001123291.1, NP_001123654.1, NP_001123655.1,NP_001123989.1, NP_001124197.1, NP_001124321.1, NP_001124322.1,NP_001124384.1, NP_001128527.1, NP_001128711.1, NP_001129171.1,NP_001129172.1, NP_001129173.1, NP_001129174.1, NP_001129293.1,NP_001129294.1, NP_001129333.1, NP_001129487.1, NP_001129511.1,NP_001135827.1, NP_001135828.1, NP_001135917.1, NP_001135918.1,NP_001136336.2, NP_001137359.1, NP_001137430.1, NP_001137457.1,NP_001138404.1, NP_001138436.1, NP_001138437.1, NP_001138438.1,NP_001138439.1, NP_001138440.1, NP_001138441.1, NP_001138442.1,NP_001138443.1, NP_001138868.1, NP_001138872.1, NP_001139280.1,NP_001139281.1, NP_001139411.1, NP_001139412.1, NP_001139501.1,NP_001139502.1, NP_001139508.2, NP_001139509.1, NP_001139748.1,NP_001139786.1, NP_001144.1, NP_001145.1, NP_001146.2, NP_001147.1,NP_001148.1, NP_001152759.1, NP_001153705.1, NP_001153706.1,NP_001154840.1, NP_001155059.1, NP_001155238.1, NP_001155901.1,NP_001156467.1, NP_001156852.1, NP_001157565.1, NP_001157566.1,NP_001157567.1, NP_001158095.1, NP_001158302.1, NP_001158886.1,NP_001158887.1, NP_001158888.1, NP_001159418.1, NP_001159477.1,NP_001159478.1, NP_001159491.1, NP_001159528.1, NP_001159529.1,NP_001159583.1, NP_001159584.1, NP_001159585.1, NP_001159586.1,NP_001159588.1, NP_001159756.1, NP_001159757.1, NP_001159897.1,NP_001159932.1, NP_001159933.1, NP_001159934.1, NP_001159968.1,NP_001160158.1, NP_001160159.1, NP_001160160.1, NP_001161694.1,NP_001161971.1, NP_001165131.1, NP_001165132.1, NP_001165901.1,NP_001165902.1, NP_001165906.1, NP_001167568.1, NP_001170775.1,NP_001171588.1, NP_001171589.1, NP_001171649.1, NP_001171650.1,NP_001171651.1, NP_001171675.1, NP_001171676.1, NP_001171677.1,NP_001171725.1, NP_001171983.1, NP_001171984.1, NP_001172006.1,NP_001172007.1, NP_001177645.1, NP_001177736.1, NP_001177931.1,NP_001177932.1, NP_001177966.1, NP_001177989.1, NP_001177990.1,NP_001180262.1, NP_001180446.1, NP_001180473.1, NP_001182016.1,NP_001182031.1, NP_001182032.1, NP_001182033.1, NP_001184044.1,NP_001185739.1, NP_001185771.1, NP_001185797.1, NP_001185798.1,NP_001185883.1, NP_001186040.1, NP_001186041.1, NP_001186054.1,NP_001186092.1, NP_001186701.1, NP_001186702.1, NP_001186703.1,NP_001186883.1, NP_001188412.1, NP_001189342.1, NP_001189343.1,NP_001189360.1, NP_001191331.1, NP_001191382.1, NP_001191439.1,NP_001192176.1, NP_001192177.1, NP_001192179.1, NP_001192180.1,NP_001193469.1, NP_001193665.1, NP_001229573.1, NP_001229753.1,NP_001229754.1, NP_001229791.1, NP_001230199.1, NP_001230200.1,NP_001230587.1, NP_001230588.1, NP_001230647.1, NP_001230648.1,NP_001230865.1, NP_001230869.1, NP_001230870.1, NP_001230871.1,NP_001230889.1, NP_001231367.1, NP_001231653.1, NP_001231867.1,NP_001238846.1, NP_001238847.1, NP_001238850.1, NP_001238851.1,NP_001238965.1, NP_001238966.1, NP_001238968.1, NP_001238978.1,NP_001238979.1, NP_001239007.1, NP_001239008.1, NP_001240752.1,NP_001241682.1, NP_001242941.1, NP_001243024.1, NP_001243064.1,NP_001243121.1, NP_001243513.1, NP_001243572.1, NP_001243573.1,NP_001243615.1, NP_001243650.1, NP_001243692.1, NP_001243728.1,NP_001243838.1, NP_001244.1, NP_001244126.1, NP_001244127.1,NP_001244305.1, NP_001244315.1, NP_001244326.1, NP_001244327.1,NP_001244328.1, NP_001244919.1, NP_001244928.1, NP_001244955.1,NP_001244957.1, NP_001245217.1, NP_001245218.1, NP_001247421.1,NP_001247422.1, NP_001247423.1, NP_001247424.1, NP_001247425.1,NP_001248341.1, NP_001248342.1, NP_001252518.1, NP_001252519.1,NP_001252520.1, NP_001254485.1, NP_001254486.1, NP_001254487.1,NP_001254488.1, NP_001257291.1, NP_001257292.1, NP_001257356.1,NP_001257411.1, NP_001257449.1, NP_001257881.1, NP_001257904.1,NP_001257905.1, NP_001257906.1, NP_001257907.1, NP_001258522.1,NP_001258670.1, NP_001258671.1, NP_001258708.1, NP_001258709.1,NP_001258710.1, NP_001258898.1, NP_001258899.1, NP_001258900.1,NP_001259025.1, NP_001263218.1, NP_001263219.1, NP_001263249.1,NP_001263382.1, NP_001264693.1, NP_001265118.1, NP_001265120.1,NP_001265121.1, NP_001265122.1, NP_001265123.1, NP_001265189.1,NP_001265190.1, NP_001265191.1, NP_001265192.1, NP_001265193.1,NP_001265194.1, NP_001265195.1, NP_001265228.1, NP_001265336.1,NP_001265337.1, NP_001265338.1, NP_001265362.1, NP_001265443.1,NP_001265543.1, NP_001265568.1, NP_001265637.1, NP_001265638.1,NP_001265641.1, NP_001268457.1, NP_001269091.1, NP_001269098.1,NP_001269153.1, NP_001269332.1, NP_001269333.1, NP_001269374.1,NP_001269378.1, NP_001269505.1, NP_001269508.1, NP_001269546.1,NP_001269547.1, NP_001269548.1, NP_001269549.1, NP_001269581.1,NP_001269582.1, NP_001269636.1, NP_001269781.1, NP_001269782.1,NP_001269783.1, NP_001269836.1, NP_001269838.1, NP_001269879.1,NP_001269881.1, NP_001269882.1, NP_001269884.1, NP_001269885.1,NP_001269961.1, NP_001273063.1, NP_001273102.1, NP_001273103.1,NP_001273104.1, NP_001273105.1, NP_001273106.1, NP_001273162.1,NP_001273163.1, NP_001273164.1, NP_001273165.1, NP_001273166.1,NP_001273178.1, NP_001273300.1, NP_001273301.1, NP_001273659.1,NP_001273718.1, NP_001273758.1, NP_001273939.1, NP_001274522.1,NP_001274523.1, NP_001275508.1, NP_001275509.1, NP_001275510.1,NP_001275582.1, NP_001276032.1, NP_001276033.1, NP_001276062.1,NP_001276674.1, NP_001276675.1, NP_001276840.1, NP_001276977.1,NP_001277151.1, NP_001277403.1, NP_001278825.1, NP_001278977.1,NP_001279.2, NP_001280014.1, NP_001280241.1, NP_001287843.1,NP_001287910.1, NP_001288169.1, NP_001288170.1, NP_001288171.1,NP_001288172.1, NP_001288758.1, NP_001288759.1, NP_001289546.1,NP_001289550.1, NP_001289551.1, NP_001289745.1, NP_001289746.1,NP_001290182.1, NP_001290203.1, NP_001290204.1, NP_001291278.1,NP_001291380.1, NP_001291381.1, NP_001291391.1, NP_001291392.1,NP_001291394.1, NP_001291395.1, NP_001291688.1, NP_001291689.1,NP_001293008.1, NP_001293013.1, NP_001293069.1, NP_001293083.1,NP_001293084.1, NP_001295030.1, NP_001295119.1, NP_001295182.1,NP_001296360.1, NP_001296769.1, NP_001298131.1, NP_001300893.1,NP_001300894.1, NP_001302466.1, NP_001303270.1, NP_001303283.1,NP_001303284.1, NP_001303285.1, NP_001303286.1, NP_001303287.1,NP_001303303.1, NP_001303991.1, NP_001303992.1, NP_001303993.1,NP_001304262.1, NP_001304263.1, NP_001304674.1, NP_001304712.1,NP_001304753.1, NP_001304754.1, NP_001304755.1, NP_001304852.1,NP_001304924.1, NP_001305051.1, NP_001305150.1, NP_001305151.1,NP_001305261.1, NP_001305438.1, NP_001305439.1, NP_001305804.1,NP_001306001.1, NP_001306002.1, NP_001306013.1, NP_001306045.1,NP_001306126.1, NP_001306127.1, NP_001306130.1, NP_001307.2,NP_001307269.1, NP_001307271.1, NP_001307508.1, NP_001307548.1,NP_001307627.1, NP_001307629.1, NP_001307631.1, NP_001307760.1,NP_001308022.1, NP_001308120.1, NP_001308296.1, NP_001308297.1,NP_001308298.1, NP_001308967.1, NP_001308968.1, NP_001308969.1,NP_001308970.1, NP_001309013.1, NP_001309014.1, NP_001309172.1,NP_001309423.1, NP_001309424.1, NP_001310245.1, NP_001310246.1,NP_001310307.1, NP_001310308.1, NP_001310309.1, NP_001310310.1,NP_001310311.1, NP_001310312.1, NP_001310313.1, NP_001310314.1,NP_001310316.1, NP_001310317.1, NP_001310318.1, NP_001310319.1,NP_001310320.1, NP_001310321.1, NP_001310322.1, NP_001310323.1,NP_001310324.1, NP_001310325.1, NP_001310326.1, NP_001310327.1,NP_001310328.1, NP_001310329.1, NP_001310330.1, NP_001310337.1,NP_001310447.1, NP_001311057.1, NP_001313507.1, NP_001313508.1,NP_001313509.1, NP_001316501.1, NP_001316838.1, NP_001316839.1,NP_001316840.1, NP_001316991.1, NP_001316992.1, NP_001317092.1,NP_001317099.1, NP_001317141.1, NP_001317145.1, NP_001317186.1,NP_001317199.1, NP_001317200.1, NP_001317273.1, NP_001317275.1,NP_001317280.1, NP_001317389.1, NP_001317390.1, NP_001317517.1,NP_001317541.1, NP_001317605.1, NP_001317656.1, NP_001317660.1,NP_001317706.1, NP_001317959.1, NP_001340.2, NP_001346.1, NP_001348.2,NP_001395.1, NP_001406.1, NP_001407.1, NP_001419.1, NP_001485.2,NP_001489.1, NP_001531.1, NP_001535.1, NP_001605.1, NP_001608.1,NP_001649.1, NP_001654.1, NP_001670.1, NP_001673.2, NP_001675.3,NP_001681.2, NP_001684.2, NP_001687.1, NP_001691.1, NP_001715.1,NP_001716.2, NP_001719.2, NP_001729.1, NP_001737.1, NP_001743.1,NP_001748.1, NP_001753.1, NP_001768.1, NP_001782.1, NP_001867.2,NP_001883.4, NP_001902.1, NP_001905.1, NP_001914.3, NP_001952.1,NP_001963.1, NP_001966.1, NP_001969.2, NP_001975.1, NP_001995.1,NP_002005.1, NP_002027.2, NP_002037.2, NP_002052.1, NP_002055.1,NP_002058.2, NP_002061.1, NP_002063.2, NP_002070.1, NP_002090.4,NP_002092.1, NP_002094.2, NP_002145.3, NP_002146.2, NP_002147.2,NP_002256.2, NP_002287.2, NP_002289.2, NP_002291.1, NP_002297.2,NP_002299.2, NP_002405.1, NP_002427.1, NP_002435.1, NP_002464.1,NP_002474.4, NP_002515.1, NP_002558.1, NP_002563.1, NP_002564.1,NP_002565.1, NP_002617.3, NP_002620.1, NP_002622.2, NP_002700.1,NP_002706.1, NP_002717.3, NP_002721.1, NP_002725.1, NP_002736.3,NP_002755.1, NP_002757.2, NP_002758.1, NP_002777.1, NP_002778.1,NP_002779.1, NP_002780.1, NP_002781.2, NP_002783.1, NP_002784.1,NP_002785.1, NP_002786.2, NP_002787.2, NP_002788.1, NP_002789.1,NP_002790.1, NP_002793.2, NP_002794.1, NP_002796.4, NP_002797.3,NP_002798.2, NP_002799.3, NP_002800.2, NP_002801.1, NP_002802.2,NP_002806.2, NP_002807.1, NP_002808.3, NP_002856.1, NP_002859.1,NP_002860.2, NP_002865.1, NP_002875.1, NP_002877.2, NP_002897.1,NP_002930.2, NP_002952.1, NP_002955.2, NP_002956.1, NP_003013.1,NP_003042.3, NP_003095.2, NP_003117.2, NP_003266.1, NP_003282.2,NP_003290.1, NP_003304.1, NP_003320.2, NP_003325.2, NP_003339.1,NP_003352.2, NP_003379.3, NP_003391.1, NP_003395.1, NP_003396.1,NP_003397.1, NP_003464.1, NP_003469.2, NP_003472.2, NP_003486.1,NP_003509.1, NP_003513.1, NP_003514.2, NP_003516.1, NP_003517.2,NP_003529.1, NP_003530.1, NP_003531.1, NP_003532.1, NP_003533.1,NP_003534.1, NP_003535.1, NP_003536.1, NP_003537.1, NP_003539.1,NP_003560.2, NP_003565.4, NP_003581.1, NP_003603.1, NP_003604.3,NP_003618.1, NP_003644.2, NP_003698.1, NP_003711.1, NP_003741.1,NP_003817.1, NP_003851.1, NP_003899.2, NP_003900.1, NP_003906.2,NP_003923.2, NP_003935.2, NP_004025.1, NP_004030.1, NP_004035.2,NP_004085.1, NP_004090.4, NP_004095.4, NP_004121.2, NP_004152.1,NP_004153.2, NP_004175.2, NP_004209.2, NP_004227.1, NP_004277.2,NP_004291.1, NP_004299.1, NP_004300.1, NP_004334.1, NP_004336.3,NP_004420.1, NP_004428.1, NP_004449.1, NP_004466.2, NP_004574.2,NP_004595.2, NP_004604.2, NP_004628.4, NP_004703.1, NP_004777.1,NP_004818.2, NP_004823.1, NP_004850.1, NP_004883.3, NP_004888.2,NP_004896.1, NP_004921.1, NP_004936.2, NP_004960.2, NP_004976.2,NP_004985.2, NP_005013.1, NP_005019.2, NP_005038.1, NP_005044.1,NP_005172.1, NP_005177.2, NP_005208.1, NP_005304.3, NP_005317.2,NP_005322.1, NP_005323.1, NP_005331.1, NP_005336.3, NP_005337.2,NP_005338.1, NP_005339.3, NP_005361.2, NP_005393.2, NP_005462.1,NP_005498.1, NP_005546.2, NP_005557.1, NP_005572.2, NP_005616.2,NP_005680.1, NP_005713.1, NP_005727.1, NP_005759.4, NP_005787.1,NP_005794.1, NP_005796.1, NP_005800.3, NP_005836.2, NP_005850.1,NP_005882.2, NP_005902.1, NP_005908.1, NP_005909.2, NP_005955.3,NP_005960.1, NP_005989.3, NP_005993.1, NP_006045.1, NP_006057.1,NP_006079.1, NP_006084.2, NP_006100.2, NP_006126.1, NP_006127.1,NP_006136.1, NP_006182.2, NP_006187.2, NP_006254.1, NP_006280.3,NP_006283.1, NP_006304.1, NP_006311.2, NP_006312.1, NP_006358.1,NP_006364.2, NP_006375.2, NP_006382.1, NP_006391.1, NP_006398.1,NP_006409.3, NP_006420.1, NP_006421.2, NP_006422.1, NP_006487.1,NP_006494.1, NP_006507.2, NP_006546.1, NP_006576.2, NP_006588.1,NP_006639.3, NP_006657.1, NP_006693.3, NP_006695.1, NP_006699.2,NP_006746.1, NP_006750.3, NP_006752.1, NP_006808.1, NP_006810.1,NP_006817.1, NP_006818.3, NP_006828.2, NP_006868.3, NP_008839.2,NP_008846.2, NP_008996.1, NP_009005.1, NP_009030.1, NP_009057.1,NP_009193.2, NP_009200.2, NP_015565.1, NP_031381.2, NP_033665.1,NP_036205.1, NP_036220.1, NP_036286.2, NP_036311.3, NP_036335.1,NP_036365.1, NP_036457.1, NP_036524.1, NP_036525.1, NP_036557.1,NP_036611.2, NP_037364.1, NP_037377.1, NP_037506.2, NP_054735.3,NP_054888.2, NP_054891.2, NP_055018.2, NP_055063.1, NP_055131.2,NP_055135.1, NP_055176.1, NP_055268.1, NP_055400.1, NP_055461.1,NP_055490.4, NP_055576.2, NP_055596.3, NP_055617.1, NP_055622.3,NP_055629.1, NP_055638.2, NP_055746.3, NP_055807.1, NP_055814.1,NP_055851.1, NP_055866.1, NP_055955.1, NP_056060.2, NP_056071.2,NP_056092.2, NP_056107.1, NP_056121.2, NP_056193.2, NP_056274.3,NP_056358.1, NP_056425.1, NP_056461.1, NP_056646.1, NP_056949.4,NP_057038.2, NP_057070.3, NP_057071.2, NP_057087.2, NP_057159.2,NP_057163.1, NP_057190.2, NP_057215.3, NP_057226.1, NP_057227.2,NP_057231.1, NP_057256.2, NP_057292.1, NP_057373.1, NP_057403.1,NP_057406.2, NP_057460.3, NP_057569.2, NP_057707.3, NP_057717.1,NP_058131.1, NP_058642.1, NP_059516.2, NP_059522.1, NP_059980.2,NP_060342.2, NP_060357.1, NP_060468.2, NP_060522.3, NP_060555.2,NP_060626.2, NP_060637.1, NP_060705.2, NP_060717.1, NP_060760.2,NP_060810.2, NP_060895.1, NP_060918.2, NP_061036.3, NP_061072.3,NP_061327.2, NP_061485.1, NP_061985.2, NP_062427.1, NP_064505.1,NP_064554.3, NP_064623.2, NP_064711.1, NP_065086.2, NP_065095.2,NP_065145.2, NP_065147.1, NP_065208.2, NP_065209.2, NP_065210.2,NP_065211.2, NP_065213.2, NP_065691.2, NP_065816.2, NP_066569.1,NP_066932.1, NP_066949.2, NP_066952.1, NP_066953.1, NP_067017.2,NP_068596.2, NP_068751.4, NP_068803.1, NP_071347.2, NP_071349.3,NP_071441.1, NP_071738.1, NP_071933.2, NP_073567.1, NP_073742.2,NP_073744.2, NP_075266.1, NP_075566.2, NP_077007.1, NP_077307.2,NP_078850.3, NP_078867.2, NP_078958.2, NP_078974.1, NP_079005.3,NP_079119.3, NP_109587.1, NP_109591.1, NP_110379.2, NP_112243.1,NP_113584.3, NP_113618.2, NP_113657.1, NP_113659.3, NP_115501.2,NP_115618.3, NP_115661.1, NP_115717.3, NP_115788.1, NP_115797.1,NP_116139.1, NP_116235.2, NP_116251.4, NP_149101.1, NP_149124.3,NP_203524.1, NP_426359.1, NP_463460.1, NP_536350.2, NP_536351.1,NP_536856.2, NP_563578.2, NP_569057.1, NP_570603.2, NP_619639.3,NP_620164.1, NP_620407.1, NP_631913.3, NP_647539.1, NP_653164.2,NP_653179.1, NP_653296.2, NP_659449.3, NP_660202.3, NP_663723.1,NP_663782.2, NP_665875.1, NP_665876.1, NP_683691.1, NP_683725.1,NP_683877.1, NP_687033.1, NP_689476.2, NP_689681.2, NP_689937.2,NP_690610.1, NP_690611.1, NP_694546.1, NP_694881.1, NP_695012.1,NP_697021.1, NP_699160.2, NP_703150.1, NP_705935.1, NP_733842.2,NP_775853.2, NP_775871.2, NP_776049.1, NP_777360.1, NP_777637.1,NP_778224.1, NP_780775.1, NP_787128.2, NP_789782.1, NP_848537.1,NP_859047.1, NP_859048.1, NP_859049.2, NP_870986.1, NP_878255.1,NP_898880.1, NP_919415.2, NP_919424.1, NP_937792.2, NP_937818.1,NP_937837.1, NP_938148.1, NP_938149.2, NP_940818.1, NP_940991.1,NP_940992.1, NP_940993.1, NP_942088.1, NP_942127.1, NP_942599.1,NP_945189.1, NP_954592.1, NP_954655.1, NP_955472.1, NP_958831.1,NP_958832.1, NP_958833.1, NP_958842.1, NP_976217.1, NP_976317.1,NP_976318.1, NP_976319.1, NP_976320.1, NP_976321.1, NP_976322.1,NP_976323.1, NP_982257.1, NP_996759.1, NP_997401.1, NP_997636.1,NP_997637.1, NP_998810.1, NP_998811.1, XP_005245019.1, XP_005245020.1,XP_005245818.1, XP_005245821.1, XP_005246799.1, XP_005247711.1,XP_005247712.1, XP_005248837.1, XP_005248935.1, XP_005248936.1,XP_005248937.1, XP_005248938.1, XP_005248939.1, XP_005249832.1,XP_005251108.2, XP_005251118.1, XP_005251120.1, XP_005251150.1,XP_005252198.1, XP_005253372.1, XP_005253374.1, XP_005253751.1,XP_005254140.1, XP_005254592.1, XP_005255286.1, XP_005255345.1,XP_005256782.1, 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XP_016883197.1, XP_016883198.1,XP_016883528.1, XP_016884292.1, XP_016884293.1, XP_016884294.1,XP_016884295.1, XP_016885269.1, XP_016885270.1, or any combinationthereof.

In some embodiments, the preparation of isolated EEV described hereinmay be obtained after inducing the production of EEV from activatederythrocytes in the blood sample of the subject. Erythrocytes may beactivated to induce EEV production, for example, using a calciumionophore (e.g., A23187), cold-storage, or ATP depletion (Prudent etal., 2015).

In some embodiments, the methods described herein may comprise a step ofremoving or depleting hemoglobin from a preparation of EEV prior todetermining the level of expression of EEV proteins. Given thesignificant amounts of hemoglobin within erythrocytes that could maskthe true nature of the protein signature in EEV, this step may improvethe detection of non-hemoglobin EEV proteins. In some embodiments,hemoglobin-depleted and hemoglobin-rich fractions may be prepared inparallel and analyzed separated for expression of EEV proteins (e.g., inmass spectrometry-based proteomic detection techniques).

In some aspects, the methods described herein may further comprisecomparing the level of an EEV protein biomarker to a suitable referencevalue indicative of the presence, stage and/or progression ofParkinson's disease, thereby clinically assessing Parkinson's disease inthe subject.

As used herein, the expression “reference value” means a control valueor range of values corresponding to a known level or range of an EEVprotein biomarker associated with the presence, stage and/or progressionof Parkinson's disease. In some embodiments, for example where theexpression level of an EEV protein biomarker has previously beenmeasured in a blood sample from a subject, the reference value may be avalue corresponding to the same subject's previous reading (e.g., abaseline). The term “suitable” in the expression “suitable referencevalue” reflects the observations reported herein that the number of EEV(and/or the protein expressed therein) in blood samples from PD subjectsmay vary depending on, for example, factors which may also affect the EVand/or EEV levels. For example, it is reported herein that a subject'sEEV levels may be affected by whether or not the subject is beingtreated for their PD symptoms, whether the subject has or previously hadcancer, whether the subject has or previously had diabetes, or whetherthe subject is taking anti-inflammatory medication.

In some embodiments, the present description relates to a method fortreating a subject with PD, the method comprising clinically assessingParkinson's disease in the subject by a method described herein, andcommencing, administering, and/or modifying PD treatment based on theclinical assessment.

The scope of the claims should not be limited by the particularembodiments set forth in the examples, but should be given the broadestinterpretation consistent with the description as a whole

EXAMPLES Example 1— Methods 1.1 Participant Recruitment and EthicStatement

Human blood was obtained from two cohorts of participants. The firstcohort was composed of Parkinson's disease (PD) patients and healthyage- and sex-matched Controls, and the second cohort was composed ofHuntington's disease (HD) patients and healthy age- and sex-matchedControls. The demographics for both cohorts are shown in Table 1. Forthe two cohorts, the Controls were recruited amongst the caregivers,spouses, family and friends of the patients. Institutional review boardsapproved this study (CHU de Québec-Université Laval, #A13-2-1096; CHUM,#14.228; Cambridge Central Regional Ethics Committee, REC #03/303 &#08/H0306/26; and Cambridge University Hospitals Foundation TrustResearch and Development department, R&D #A085170 & A091246) inaccordance with the Declaration of Helsinki, and written informedconsent was obtained from all participants.

In the case of PD patients, the clinical evaluation included measures onthe Unified Parkinson Disease Rating Scale (UPDRS), the Hoehn and Yahr(H&Y) stage, the Mini Mental State Examination (MMSE), the Addenbrooke'sCognitive Examination (ACE), and the Beck Depression Inventory (BDI). Inthe case of the HD patients, we collected their scores on the UnifiedHuntington Disease Rating Scale (UPDRS), Total Functional capacity (TFC)and calculated values for burden of disease (BDS). All the clinicalevaluations were conducted within 9 months of the blood drive.Participants were further asked to fill out a questionnaire related tohealth issues and medication and their full blood count performed on theday of blood sampling.

Of note, participants excluded from the present EEV-related analysesincluded those with diabetes and those suffering or having suffered fromcancer, because we observed a significant PD-independent increase in EEVconcentration in the platelet-free plasma of these participants.Furthermore, PFP samples with elevated free hemoglobin (>45 000 ng/mL),potentially due to hemolysis at blood sampling, were also excluded fromEEV-related analyses, which explains the discrepancies between the totalnumber of participants initially recruited and those contained in eachanalysis.

TABLE 1 Participant demographics Table 1: Participant clinicalinformation. Parkinson's disease (PD) cohort PD Patients - Stages ofdisease Ctrl Unknown Mild Moderate Severe P value n 37 7 12 33 8 Age66.8 69.8 66.7 71.1 75.0* 0.04 Gender F (M) 18 (19) 1 (6) 6 (6) 16 (17)0 (8) 0.05 Disease severity Hoehn & Yahr (n) 1 ± 0.3 (12) 2 ± 0.2 (33) 3± 0.5 (8) <0.0001 UPDRS (n) 38 ± 11 (6) 52 ± 19 (17) 73 ± 20 (6) 0.02ACE (n) 96 ± 4 (6) 92 ± 7 (17) 84 ± 14 (6) 0.13 MMSE (n) 29 ± 2 (7) 29 ±1 (19) 26 ± 3 (6) 0.01 BDI (n) 3 ± 2 (6) 4 ± 2 (17) 13 ± 7 (4) 0.03Comorbidities Asthma 3 1 1 5 0 0.71 Hypertension 10 1 2 10 3 0.76Diabetes 2 0 0 1 2 0.10 Cancer 5 0 3 4 1 0.64 Allergies 2 0 2 6 2 0.28Depression 3 1 2 1 2 0.29 Hypercholesterolemia 5 0 1 6 1 0.73Huntington's disease (HD) cohort HD Patients - Stages of disease CtrlPre-HD Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 P value n 55 11 15 13 1210 2 Age 55.0 37.5 53.1 54.2 58.3 58.1 55.5 0.02 Gender F (M) 31 (22) 6(5) 5 (10) 4 (9) 8 (4) 7 (3) 1 (1) 0.26 Disease severity UHDRS (n) 2.7(11) 15.7 (14) 34.5 (11) 42.9 (12) 55.9 (10) 67.5 (2) <0.001 TFC (n) 13(16) 13 (11) 12.5 (15) 7.8 (13) 4.3 (12) 1.6 (10) 0 (2) <0.001 CAG (n)28.3 (3) 41.1 (10) 42.3 (13) 42.6 (12) 43.7 (7) 44.3 (7) <0.001 BDS (n)206 (10) 337 (13) 356 (12) 442 (7) 465 (7) <0.001 Comorbidities Asthma 00 1 0 0 0 0 0.65 Hypertension 4 1 2 1 1 2 0 0.92 Diabetes 3 1 1 1 1 1 00.99 Cancer 0 0 0 0 0 0 0 Allergies 3 0 2 2 0 0 0 0.33 Depression 8 1 13 6 4 1 0.0497 Hypercholesterolemia 8 1 1 0 0 1 0 0.32 (PD cohort)Disease severity levels in relation to the H&Y scale (score): Mild(1-1.5); Moderate (2-2.5); Severe (3-3.5). *p < 0.05 vs. CTRL.Statistical analyses were performed using a Welch ANOVA followed byDunnett's multiple comparison test. Disease severity was evaluatedwithin 6 months of blood sampling. Comorbidities were determined frommedical information reported by the participant or caregiver. Cancerrefers to participant having suffered from cancer in the past. (HDcohort) Disease severity levels in relation to the TFC scale: Stage 1(11-13); Stage 2 (7-10); Stage 3 (3-6); Stage 4 (1-2); Stage 5 (0).Disease severity was evaluated within 6 months of blood sampling.Comorbidities were determined from medical information reported by theparticipant or caregiver. Abbreviations: ACE, Addenbrooke's cognitiveexamination; BDI, Beck depression inventory; BDS, Burden of DiseaseScore; CAG, Trinucleotide repeat; MMSE, Mini-Mental State Examination;UHDRS, Unified Huntington's Disease Rating Scale; TFC, Total FunctionCapacity.

1.2 Preparation of Platelet-Free Plasma (PFP) and Extracellular Vesicle(EV) Labeling

Citrated blood was centrifuged twice for 15 minutes at 2500 g at roomtemperature. Platelet-free plasma (PFP) was harvested and stored at −80°C. within 2 hours of collection following guidelines suggested byLacroix and colleagues (Lacroix et al., 2012).

For all experiments, diluted annexin-V buffer (BD Pharmingen,Mississauga, ON, Canada) and phosphate buffered saline (PBS) werefiltered on 0.22 μm pore size membranes. To quantify the EV according totheir cell of origin, the following surface markers were used: CD235a+(erythrocytes) (5 μL), CD31+/CD41− (endothelial cells) (1 μL), CD41+(platelets) (5 μL), CD45+ (leukocytes) (3 μL), CD45+CD14+ (monocytes)(10 μL), and CD45+CD15+ (granulocytes) (2 μL), with or without annexin-Vstaining (5 μL). PFP (5 μL) was incubated withPhenylalanyl-prolyl-arginyl Chloromethyl Ketone (PPACK) (Calbiochem,Etobicoke, ON, Canada) for 5 minutes, followed by a 30-minute incubationwith antibodies and annexin-V in a final PBS volume of 100 μL, all atroom temperature. Finally, the samples were diluted to a final volume of2 mL prior to FACS analysis. The following antibodies were purchased atBD Pharmingen and used throughout the experiments: FITC-conjugated mouseanti-human CD235a (clone GA-R2 (HIR2), 1/20), PE-conjugated mouseanti-human CD31 (clone WM59, 1/100), V450-conjugated mouse anti-humanCD41a (clone HIPS, 1/20), APC mouse anti-human CD14 (clone M5E2, 1/10),PE-conjugated mouse anti-human CD15 (clone H198, 1/50), V450-conjugatedmouse anti-human CD45 (clone H130, 1/33), V450- andPerCP-Cy™5.5-conjugated annexin-V (1/33 and 1/10, respectively).

1.3 Flow Cytometry Quantification

For EV quantification, we used a FACS Canto II Special Order ResearchProduct equipped with a forward scatter (FSC) coupled to aphotomultiplier tube (FSC-PMT). Flow cytometer performance tracking wascarried out daily using the BD cytometer setup and tracking beads (BDBiosciences, San Jose, CA, USA). The size of the EV was determined usingfluorescent silicone beads of 100, 500 and 1000 nm. Controls andoptimization of the detection method are presented in FIGS. 1A-1F. Thesettings for the EV detection were determined as described previously(Rousseau et al., 2015) using a threshold of 200 for SSC. Between PD andHD analyses, the blue laser had to be replaced for maintenance issuesand therefore laser settings were reassessed. For FSC-PMT, the assignedvoltage was 363 (PD) and 160 (HD) Volts. For SSC, the assigned voltagewas 407 (PD) and 300 (HD) Volts. All other parameters were set between450 and 500 Volts. The acquisition of EV was performed at low speed withan approximate rate of 10 μL/min. To determine background noise level,antibody mixes were incubated in absence of PFP sample and unlabeled PFPwas used as a negative control.

1.4 Statistical Analyses

All statistical analyses pertaining to Examples 2.1 and 2.2 wereperformed using “The Statistics and Machine Learning Toolbox” providedby MathWorks™ under the MATLAB™ platform. The version used wasMATLAB®R2015a. The analysis included the scatter plot, the classicalleast-squares linear regression model, the R-squared and p values, aswell as Pearson's goodness-of-fit model. Interval cut-off values weredetermined using a loop program developed in MATLAB™. Model diagnostics,including residual behaviour and homoscedastivity, were also obtainedwith the same Toolbox.

1.5 Production and Purification of EEV

Blood was collected in heparin tubes and centrifuged for 10 minutes at282 g at room temperature. Blood cells were washed first in PBS-2% FBS,then with 0.9% sodium chloride solution and centrifuged for 10 minutesat 750 g. To avoid leukocyte and/or platelet contamination, the buffycoat and the upper fraction of erythrocytes were removed. To preserveerythrocytes, two volumes of glycerolyte 57 solution (57% glycerol, 142mM sodium lactate, 1 mM KCl, 25 mM sodium phosphate pH 6.8) were addedto the pellet and stored at −80° C.

For the production of EEV, red bloods cells were thawed and EVproduction was induced as previously described (Minetti et al., 2004).Briefly, the erythrocyte pellet was activated with 3 volumes of calciumionophore solution (150 mM NaCl; 10 mM Tris-HCl; 1 mM CaCl₂; 5 μMionophore A23187 (Sigma, St Louis, MO)) for 30 minutes at 37° C. Theactivation was stopped by the addition of 5 mM EDTA. Remainingerythrocytes were pelleted at 15 000 g for 20 minutes. The EEV werecentrifuged at 20 000 g for 90 minutes and washed once in PBS. The EEVpellet was resuspended in PBS and frozen at −80° C. until furtheranalyses.

1.6 C-Reactive Protein, Free Hemoglobin and α-Synuclein Quantification

The concentrations of C-reactive protein (CRP) and free hemoglobin weredetermined in the PFP of all donors using the RayBio™ Human CRP ELISAKit (RayBiotech, Norcross, GA, USA) and the Hemoglobin Human ELISA kit(Abcam, Toronto, ON, Canada). To quantify α-synuclein (α-Syn) inerythrocytes and EEV, we used the human α-Syn ELISA kit (ThermoFisherScientific, Waltham, MA, USA). Absorbance values were measured at 450 nmusing a multi-detection microplate reader (Synergy HT; BioTek; Winooski,VT, USA). All ELISA tests were performed according to the manufacturer'sinstructions.

1.7 Scanning Electron Microscopy

Preparations of erythrocytes (5 μL) were fixed in 2% paraformaldehydeand 2.5% glutaraldehyde in PBS buffer at least 24 hours before standarddehydration. Samples were washed 3 times for 10 minutes with sodiumcacodylate buffer (0.1 M, pH 7.3) and fixed with 1% osmium tetroxide insodium cacodylate buffer for 90 minutes. Subsequently, samples werewashed and processed in 50%, 70%, 90% and 100% ethanol for dehydration(10 minutes/step). Finally, samples were soaked in two subsequent bathsof 100% ethanol, for 40 minutes and 10 minutes, air-dried overnight andcoated with palladium. Observations were completed using a JEOL 6360LVscanning electron microscope (JEOL, Peabody, MA, USA).

1.8 Transmission Electron Microscopy

Preparations of EEV (30 μL) and activated erythrocytes (5 μL) were fixedin 2% paraformaldehyde at least 24 hours before being dehydrated andsealed in LR white resin. Slices of LR white resin were placed on aFormvar/carbon-coated grid and processed for immunolabeling. The tissuesmounted on grids were blocked in 0.5% BSA-c (Aurion, Wageningen, TheNetherlands) in HBSS and incubated for 120 minutes with rabbitanti-α-Syn antibody (Abcam, Toronto, ON, Canada) or rabbit anti-α-Syn(phospho S129) antibody (Abcam, Toronto, ON, Canada), both diluted at1:250 in HBSS and washed several times with distilled water. Finally,the grids were incubated for 60 minutes with an anti-rabbit IgGconjugated to 6 nm gold particles (EMS, Hatfield, PA, USA) diluted at1:200 and washed several times with distilled water to ultimately befixed in 2.5% glutaraldehyde (EMS, Hatfield, PA, USA) in HBSS for 15minutes. For this last step, the grids were treated with 3% uranylacetate-0.075 M oxalate (pH 7.0) (EMS, Hatfield, PA, USA) for 1 minute,which was followed by several washes in distilled water. Observationswere completed with a TECNAI Spirit G2 transmission electron microscopeat 80 kV (FEI, Hillsboro, OR, USA).

1.9 Mass Spectrometry Analysis and Label Free Protein Quantification

For proteomic analyses, EEV from 4 individuals per group (Control, mildPD and moderate PD) were prepared as described above. For eachindividual, 25 μg of protein sample, according to Bradford proteinassay, were migrated onto an electrophoresis gel 4-12% Bis-Tris toseparate hemoglobin from higher proteins. Following gel staining usingSypro Ruby (Thermo Fischer Scientific), the 12 kDa band corresponding tothe hemoglobin size was cut out and the remaining part of the gelfurther fractioned into 7 slices, exposed to trypsin digestion andpeptide extraction on a MassPrep™ liquid handling robot (Waters,Milford, USA) according to the manufacturer's specifications and to theprotocol of Shevchenko et al., 1996, with the modifications suggested byHavlis et al., 2003. The extracted peptides from the 7 slices of thesame individual were pooled and analyzed by nanoLC-MS/MS. The excisedhemoglobin gel slices were also analyzed in the same conditions. One μgof each individual sample was injected on a Dionex UltiMate™ 3000nanoRSLC system (Thermo Scientific) equipped with a nanoviper AcclaimPepmap100™, C18, 3 μm, 75 μm×column (Thermo Scientific) connected to thenanoelectrospray source of an Orbitrap Fusion™ mass spectrometer (ThermoScientific). The peptides were eluted at 300 nL/min using anacetonitrile gradient of 90 minutes and the mass spectrometer wasoperating in Data Dependent Acquisition mode. Peptide masses weremeasured in MS spectra detected in the orbitrap at 120K resolution. MSMSfragmentation spectra of peptides were generated by Higher energyCollisional Dissociation (HCD) and detected in the ion trap. Spectrawere searched against a human protein database (Uniprot CompleteProteome, taxonomy Homo sapiens—83512 sequences) using Andromeda™ searchengine included in MaxQuan™ software version 1.5.5.1 (Cox et al., 2008).MaxQuant™ was also used to validate proteins and peptides at 1% FalseDiscovery Rate using a target/decoy database search and to perform LabelFree Quantification of the identified proteins using the ‘match betweenruns’ option.

1.10 Further Statistical Analyses

For FIGS. 3C and 3D, data were first tested for normality using theD'Agostino & Pearson normality test. Comparisons between groups wereobtained by Mann-Whitney U test or Kruskal-Wallis ANOVA and performedusing Prism 6.0 (GraphPad Software, LaJolla, CA). For analyses displayedin FIG. 4C, the ‘Intensity values’ contained in the output‘proteingroup.txt’ file of MaxQuant™ were used to quantify eachidentified protein in each individual sample. The values were normalizedby the median of each column (all intensity values of proteins for onesample). The missing values were imputed with a noise valuecorresponding to the 1-percentile of each sample column. For eachcomparison between two groups (Control, mild PD or moderate PD),proteins with too many imputed values where considered not quantifiable(a minimum of three not-imputed values in one of the 2 groups arerequired). A protein ratio was calculated between the two groups usingthe average of intensity values in each group. Finally, a statisticalWelch's test was performed between the two groups. The protein ratioswere transformed into log 2(ratio) then centered by calculation of az-score (z-score=(x−μ)/σ). A protein was considered as variant if itfulfilled the following criteria: minimum of 2 peptides quantified,Welch's test p value<0.05 and absolute value of z-score>1.96(corresponding to values outside of the 95% confidence interval). TheGene Ontology enrichment analysis on the identified proteins (FIG. 4D)was performed on the Cytoscape™ platform (v. 3.4.0) using the BinGO™software version 3.0.3 (Maere et al., 20005) against all human geneswith GO annotation (Uniprot-GOA generated 2015-06-22). Enrichment wascalculated by hypergeometric test and Bonferroni Family-Wise Error Rate(FWER) was used to correct for multiple testing. The data for theresulting 8 proteins was standardized, hierarchically clustered andvisualized as a heatmap by using the statistical framework R (R CoreTeam, 2016). The robustness of the nodes was evaluated by computingApproximately Unbiased (AU) p values using the R package pvclust (10000bootstraps, average method and correlation-based dissimilarity matrix)(Suzuki et al., 2006).

Example 2— Results

The cohorts studied here included Parkinson's disease (PD) (n=60) andHuntington's disease (HD) patients (n=63) of all stages (see Example1.1), as well as their respective age- and sex-matched healthy controls(n=37; n=55, respectively). The demographics for both cohorts are shownin Table 1. Full blood counts (erythrocytes, lymphocytes, platelets,leukocytes, monocytes, neutrophils) and C-reactive protein (indicativeof an inflammatory response) quantification were obtained for allparticipants, but they did not reveal any significant differencesbetween groups (data not shown). Similarly, the hematocrit, the meancorpuscular hemoglobin, as well as the mean corpuscular volume valueswere similar between PD and control groups (data not shown).

2.1 PD Patients Exhibit a Disease-Specific Increase inErythrocyte-Derived EV

Platelet-free plasma (PFP) and extracellular vesicles (EV) were labeledand quantified according to their cell of origin for all participants,as described in Examples 1.2 and 1.3. Results are summarized in Table 2A(PD patients and controls) and Table 2B (HD patients and controls).

As shown in Table 2A, no significant differences between PD patient andcontrol samples were observed in the number of EV originating fromplatelets, endothelial cells, monocytes, granulocytes, and leukocytes.Similarly, as shown in Table 2B, no significant differences between HDpatient and control samples were observed in the concentrations of EVoriginating from these same cell types.

Interestingly, a significant increase in erythrocyte-derived EV inpatients with PD was observed, as compared to the control group (seevalues highlighted in black in Table 2A). This increase inerythrocyte-derived EV in patients with PD was disease-specific, as thesame effect was not observed in erythrocyte-derived EV in patients withHD (Table 2B).

TABLE 2A Quantification of extracellular vesicles (EV) derived fromdifferent cell types of PD patients and controls CTRL PD P Cell typeMarkers Units n Mean SEM n Mean SEM value Platelets CD41+PS− ×10³/μL 377.88 1.68 59 10.3 1.33 0.27 CD41+PS+ 37 15.2 3.20 59 17.9 2.53 0.51CD41+CD31+ 37 1.51 0.69 59 1.99 0.54 0.59 CD41+ total 37 23.1 4.62 5928.2 3.66 0.38 EV CD41+/platelet 35 0.106 0.021 57 0.125 0.016 0.49Endothelial cells CD31+CD41−PS− ×10³/μL 37 15.8 8.04 59 11.7 6.37 0.75CD31+CD41−PS+ 37 0.91 0.13 59 0.92 0.10 0.96 CD31+CD41− total 37 16.78.03 59 12.6 6.36 0.75 Monocytes CD45−CD14+ PS− ×10³/μL 37 1.70 0.30 591.62 0.24 0.85 CD45−CD14+ PS+ 37 1.20 4.00 59 5.84 3.17 0.50 CD45+CD14+PS− 37 0.16 0.04 59 0.14 0.03 0.74 CD45+CD14+PS+ 37 0.60 0.79 59 1.470.63 0.59 CD14+ total 37 3.66 4.88 59 9.06 3.87 0.60 EV CD14+/monocyte35 7.08 1.99 57 9.16 1.56 0.41 Granulocytes CD45−CD15+ PS− ×10³/μL 3712.3 7.96 59 16.7 6.30 0.92 CD45−CD15+ PS+ 37 2.21 0.77 59 1.39 0.610.47 CD45+CD15+ PS− 37 0.55 0.36 59 1.15 0.29 0.20 CD45+CD15+PS+ 37 1.010.30 59 1.25 0.24 0.56 CD15+ total 37 16.0 8.83 59 20.6 6.99 0.91 EVCD15+/granulocyte 35 3.70 0.64 57 3.16 0.50 0.53 Leukocytes CD45+ total×10³/μL 37 10.4 2.21 59 13.8 1.75 0.26 Erythrocytes CD235a+PS− ×10³/μL36 18.2 46.5 59 32.0 36.3 0.04 CD235a+PS+ 36 0.22 0.07 59 0.29 0.05 0.70CD235a+ total 36 18.4 47.0 59 32.3 36.7 0.04 EV CD235a+/erythrocyte 340.0039 0.011 57 0.0069 0.008 0.04 Abbreviations: CD235a, glycophorin A;EV, extracellular vesicle; PD, Parkinson's disease; PS,phosphatidylserine.

TABLE 2B Quantification of extracellular vesicles (EV) derived fromdifferent cell types of HD patients and controls CTRL HD pre-manifest HDP Cell type Markers Units n Mean SEM n Mean SEM n Mean SEM valuePlatelets CD41+PS− ×10³/μL 54 9.2 2.2 10 4.3 1.3 50 6.1 1.0 0.78CD41+PS+ 54 19.3 4.8 10 7.1 2.0 50 12.4 2.4 0.74 CD41+ total 54 28.4 6.910 11.4 3.2 50 18.6 3.4 0.70 EV CD41+/platelet 53 0.12 0.03 10 0.05 0.0248 0.08 0.01 0.34 Endothelial CD31+CD41−PS− ×10³/μL 54 1.4 0.3 10 0.60.2 50 1.2 0.2 0.31 cells CD31+CD41−PS+ 54 0.68 0.16 10 0.25 0.06 500.46 0.09 0.59 CD31+CD41− total 54 2.1 0.4 10 0.8 0.2 50 1.7 0.3 0.26Monocytes CD45−CD14+ PS− ×10³/μL 54 3.4 1.1 10 1.6 0.2 51 1.6 0.1 0.91CD45−CD14+ PS+ 54 1.8 0.3 10 0.8 0.3 51 1.5 0.2 0.14 CD45+CD14+ PS− 540.18 0.07 10 0.069 0.016 51 0.056 0.008 0.34 CD45+CD14+PS+ 54 0.62 0.1210 0.24 0.06 51 0.55 0.14 0.12 CD14+ total 54 6.0 1.3 10 2.6 0.4 51 3.70.4 0.08 EV CD14+/monocyte 53 12.3 2.5 10 5.7 0.6 48 8.0 1.0 0.13Granulocytes CD45−CD15+ PS− ×10³/μL 54 1.2 0.1 10 1.2 0.3 51 1.5 0.20.33 CD45−CD15+ PS+ 54 0.12 0.04 10 0.18 0.08 51 0.22 0.11 0.33CD45+CD15+ PS− 54 0.20 0.05 10 0.07 0.02 51 0.15 0.04 0.64 CD45+CD15+PS+54 0.25 0.05 10 0.13 0.06 51 0.20 0.04 0.39 CD15+ total 54 1.7 0.2 101.6 0.4 51 0.20 0.3 0.67 EV CD15+/ 53 0.41 0.04 10 0.42 0.13 48 0.500.08 0.75 granulocyte Leukocytes CD45+ total ×10³/μL 54 33.4 2.7 10 31.65.3 51 31.7 2.4 0.88 Erythrocytes CD235a+PS− ×10³/μL 54 15.2 2.0 10 10.33.5 51 14.1 1.4 0.16 CD235a+PS+ 54 1.1 0.2 10 0.4 0.2 51 1.1 0.1 0.04CD235a+ total 54 16.4 2.0 10 10.7 3.5 51 15.3 1.5 0.09 EV 54 0.00350.0005 10 0.0023 0.0008 50 0.0033 0.0003 0.11 CD235a+/erythrocyteAbbreviations: CD235a, glycophorin A; EV, extracellular vesicle; HD,Huntington's disease; PS, phosphatidylserine.

2.2 Increase in Erythrocyte-Derived EV in PD Patient Samples Correlateswith PD Progression and PD Treatment

To evaluate its suitability as a potential biomarker for monitoring PDprogression, we examined correlations between the number oferythrocyte-derived EV (EEV) and the Unified Parkinson's Disease RatingScale (UPDRS) PD staging system, because of its greater sensitivity andthe recent publications validating this approach (Martinez-Martin etal., 2015). Strikingly, statistical linear regression analysis revealedstrong correlations between the number of erythrocyte-derived EV and PDstage/progression. As shown in FIG. 2A, strong correlations(correlations exceeding were observed between the number oferythrocyte-derived EV (expressed as CD135a+ EV/total number oferythrocytes) and patient UPDRS score, and thus PD stages. The R² valuesobtained demonstrated that in both “mild” and “moderate” PD patientgroups, at least 87% of the variation in the total number ofEEV/erythrocytes is due to the variation of the UPDRS. Moreover, theresults are significant with respect to the p values obtained for eachfit, since they fall below the 5% confidence level. Hence, thestatistical tests on EEV counts uncovered a clear cut-off point betweenmild and moderate PD patients, which could not be accounted for by dailylevodopa doses administered to the patient (FIG. 2A, see details of the5 patients pinpointed).

The above correlations observed with respect to the number of EEV in PDpatients was found to be disease-specific, since a similar analysisperformed in HD patients failed to reveal the same strong correlations(see FIG. 2B). In the HD cohort, the total number of EEV betweenpre-manifest and manifest HD was similar to their age- and sex-matchedhealthy Controls. In contrast to PD, correlation analyses failed toreveal an association between the number of EEV and HD stage using theUnited Huntington's Disease Rating Scale (UHDRS) score (FIG. 2B). Basedon this, all our subsequent analyses focused only on the PD cohort. Itis should be re-emphasized that total blood counts did not indicate anysignificant differences in the number of endothelial cell-, platelet-,monocyte- and granulocyte-derived EV in the platelet-free plasma (PFP)in PD (Table 2A) and HD patients (Table 2B), when compared to theirrespective control cohorts.

We have thus identified at least two distinct groups of PD patients withhighly significant correlations to the number of EV derived fromerythrocytes, which relates to PD stage and/or PD treatment (FIG. 2A).Strikingly, these correlations appear to be specific to PD, as similarcorrelations were not observed in the cohort of HD patients (of varyingdegrees of severity) in which we performed identical analyses (FIG. 2B).

2.3 α-Syn is not Differentially Expressed in Normal Vs. Diseased PDConditions

Having established that EEV counts correlate with disease state in PD,we aimed to assess whether α-Syn—which is not only the main component ofLewy bodies but is highly expressed in most blood cells—wasdifferentially expressed in normal vs. diseased conditions. For this, weopted to use scanning electron microscopy, but this did not reveal anymorphological changes between resting and activated erythrocytes ineither condition (FIG. 3A). We further used transmission electronmicroscopy to quantify the number of EEV containing α-Syn andphosphorylated (serine 129) forms of the protein but again nosignificant differences between PD patients and age- and sex-matchedhealthy Controls (FIGS. 3B and 3C) were observed. Quantified α-Synlevels in EEV from PD patients and Controls using commercial ELISA kitscorroborated these results (FIG. 3D).

2.4 Analysis of the EEV Proteome Revealed 8 Differentially ExpressedProteins

Since our combined quantifications (ELISA and transmission electronmicroscopy) suggested that α-Syn levels could not be used as a bloodmarker of disease, we sought to obtain the specific protein signature ofEEV from mild and moderate PD patients (with respect to the UPDRSscores) and their age-matched Controls. Given the significant amounts ofhemoglobin within erythrocytes that could mask the true nature of theprotein signature in EEV, we performed a label free quantitativeproteomic analysis by nanoLC/MSMS (Wither et al., 2016) using twodistinct approaches: with and without hemoglobin.

By removing the hemoglobin, we identified a total of 818 proteins incomparison with 356 when we did not perform this methodological step(refer to Table 4 for complete list of proteins), which clearly,provides a much more thorough evaluation of the protein content of EEV.Additionally, a Gene Ontology enrichment analysis on the ‘CellularComponent’ ontology performed on the two sets of identified proteins incomparison with the whole human proteome, revealed that our samples areenriched with elements associated to ‘vesicles’ and ‘hemoglobin complex’which show the efficiency of our EEV production and purificationprotocol (data not shown).

Out of the 818 proteins identified in the proteome of EEV, 8 had theirexpression significantly modified according to the different stages ofPD (FIGS. 4A and 4B). Hierarchical clustering, coupled to a heatmap(FIG. 4D), allowed us to group individuals according to stages ofdisease (Control, mild PD and moderate PD) and provided compellingevidence that the 8 proteins identified could also be grouped into threecategories. Proteins of group I were highly and predominantly expressedin Controls, proteins of group II were highly and predominantlyexpressed in mild PD patients, and proteins belonging to group Ill werehighly and predominantly expressed in moderate PD patients (FIGS. 4B and4C; Table 3). This data set was further confirmed by volcano plots (FIG.5 ). Of note, two proteins of group I are associated with the regulationsystem of the cell (ABHD148, NADSYN1) and one protein significantlyexpressed in moderate PD patients (ATP5A1) is involved in the regulationof mitochondrial ATP.

TABLE 3 EEV proteins differentially expressed according to differentstages of PD Groups Protein Gene I Axin interactor,dorsalization-associated AIDA (Control) protein Alpha/beta hydrolasedomain-containing ABHD14B protein 14B Glutamine-dependent NAD(+)synthetase NADSYN1 II Dihydropteridine reductase QDPR (mild PD) Alcoholdehydrogenase [NADP(+)] AKR1A1 CB1 cannabinoid receptor-interactingCNRIP1 protein 1 III Ubiquitin carboxyl-terminal hydrolase USP24(moderate 24 PD) ATP synthase subunit alpha, ATP5A1 mitochondrial

Fold changes and the results of statistical analyses of the proteins ofTable 3 are shown in Table 3.1, in which the proteins are listed inorder of their “fold” difference for each group comparison. For example,a “fold” of 1.931 indicates that the AKR1A1 protein was detected in theisolated EEV preparations almost two times higher in the mild group, ascompared to the control group.

TABLE 3.1 Fold changes and statistics of EEV proteins of Table 3 ProteinGene Welch's test Fold change Z-score Mild (Group II) v. Controls (GroupI) Alcohol dehydrogenase [NADP(+)] AKR1A1 0.019 1.931 2.25 Alpha/betahydrolase domain-containing protein 14B ABHD14B 0.016 0.274 −3.57Moderate (Group III) v. Controls (Group I) ATP synthase subunit alpha,mitochondrial ATP5A1 0.028 3.906 3.25 Glutamine-dependent NAD(+)synthetase NADSYN1 0.009 0.391 −2.31 Axin interactor,dorsalization-associated protein AIDA 0.046 0.379 −2.38 Alpha/betahydrolase domain-containing protein 14B ABHD14B 0.017 0.282 −3.09Moderate (Group III) v. Mild (Group II) ATP synthase subunit alpha,mitochondrial ATP5A1 0.027 3.629 2.77 Ubiquitin carboxyl-terminalhydrolase 24 USP24 0.036 2.971 2.29 CB1 cannabinoid receptor-interactingprotein 1 CNRIP1 0.014 0.352 −2.80 Alcohol dehydrogenase [NADP(+)]AKR1A1 0.002 0.342 −2.87 Dihydropteridine reductase QDPR 0.040 0.341−2.88 Proteins were considered as significantly differentially expressedif they respected two conditions: Z-score >1.96 or <−1.96, and Welch'stest p-value < 0.05.

As seen in Table 3.1 and in FIG. 4C, the ABHD14B protein (alpha/betahydrolase domain-containing protein 14B) was detected over 3.5-foldlower both in the mild v. controls and in the moderate v. controlsgroups, suggesting that this EEV protein may be a useful biomarker forclinically assessing symptomatic PD subjects. The results in Table 3.1and in FIG. 4C suggest that the AKR1A1 protein (alcohol dehydrogenase[NADP(+)]) may be a useful biomarker for clinically assessing mild PDsubjects, as this EEV protein was detected almost 2-fold higher in mildv. control PD subjects, but was not differentially expressed in moderatev. control PD subjects. Finally, the results in Table 3.1 and in FIG. 4Csuggest that the ATP5A1 protein may be a useful biomarker for clinicallyassessing moderate PD subjects, since this protein was detected over3.6-fold higher in both moderate v. controls and in moderate v. mild PDsubjects.

Example 3— Discussion

We have identified biomarkers correlating to different states of PDbased on the quantification of EV shed from erythrocytes and UPDRSscores. In particular, we have shown a strong correlation between thenumber of EEV and the clinical expression/stages of PD. Finding thislevel of correlation in a 60-patient sample size in such a heterogeneousdisorder highlights the robustness of the biomarkers identified herein.

Although staging of PD is often done using the H & Y clinical scale, wesought to use the UPDRS given its greater sensitivity and the recentpublications validating this approach (Martinez-Martin et al., 2015).Using these scores, we found that mild PD patients—with a UPDRS scorelower than 37—are characterized by an increased number of EV during themild disease stage (correlations=0.886); and that the same patternrepeated itself with patients who had UPDRS scores between 37 and 75(correlations=0.873). When comparing individual patients from bothcorrelation curves, the dose of levodopa taken daily did not seem toaccount for the differences, as there was no correlation between thelevodopa dose and the number of EEV.

Finally, we herein report an improved method to perform proteomicanalysis of EEV in blood samples by removing hemoglobin, a large proteinthat can easily mask other ones within a protein signature. Indeed, thehigh dynamic range of protein concentrations in erythrocytes andtherefore in EEV, due to the high abundance of hemoglobin, decreases thecapacity of the mass spectrometer to detect signals corresponding to lowabundance proteins. Analyzing the hemoglobin separately from the otherproteins of other molecular weights allowed us to go deeper in the EEVproteome by identifying 129% more proteins than in the initial analysis.

TABLE 4 Proteins identified in the EEV proteome. Lists of the proteinsidentified by LC-MS/MS analysis using Andromeda ™/MaxQuant ™ searchengine in the Uniprot Complete Proteome Homo sapiens database for thenon-hemoglobin-depleted sample (356 proteins) (A), or when thehemoglobin-depleted and hemoglobin-containing fractions were analyzedseparately (818 proteins) (B). The lists were filtered at 1% FalseDiscovery Rate using a target/decoy database search. Razor + uniqueProtein ID Protein names Gene names peptides (A). UNIPROT COMPLETEPROTEOME HOMO SAPIENS DATABASE FOR THE NON-DEPLETED SAMPLE P02549Spectrin alpha chain, erythrocytic 1 SPTA1 136 P11277 Spectrin betachain, erythrocytic SPTB 123 P16157 Ankyrin-1 ANK1 70 P02730 Band 3anion transport protein SLC4A1 38 P55072 Transitional endoplasmicreticulum ATPase VCP 33 P16452 Erythrocyte membrane protein band 4.2EPB42 33 P11171 Protein 4.1 EPB41 33 P04040 Catalase CAT 32 Q13228Selenium-binding protein 1 SELENBP1 29 B4DT77 Annexin; Annexin A7 ANXA727 Q8WUM4 Programmed cell death 6-interacting protein PDCD6IP 27 P35612Beta-adducin ADD2 26 P68871 Hemoglobin subunit beta; LW-hemorphin-7;Spinorphin HBB 25 Q00610 Clathrin heavy chain 1 CLTC 25 P69905Hemoglobin subunit alpha HBA1 22 B4DVE7 Annexin A11 ANXA11 20 J3QLD9Flotillin-2 FLOT2 20 P08758 Annexin A5; Annexin ANXA5 19 P09525 AnnexinA4; Annexin ANXA4 18 O75955; Flotillin-1 FLOT1 18 P11142 Heat shockcognate 71 kDa protein HSPA8 18 P32119 Peroxiredoxin-2 PRDX2 17 P00491Purine nucleoside phosphorylase PNP 17 P27105 Erythrocyte band 7integral membrane protein STOM 17 P00918 Carbonic anhydrase 2 CA2 17P23634 Plasma membrane calcium-transporting ATPase 4 ATP2B4 17 C9JIF9Acylamino-acid-releasing enzyme APEH 17 P00915 Carbonic anhydrase 1 CA116 P63261 Actin, cytoplasmic 2 ACTG1 16 Q5VU58 Tropomyosin alpha-3 chainTPM3 16 P30041 Peroxiredoxin-6 PRDX6 16 E7EU23 Rab GDP dissociationinhibitor beta GDI2 16 Q00013 55 kDa erythrocyte membrane protein MPP116 E7EV01 Calpain-5 CAPN5 16 J3KPS3 Fructose-bisphosphate aldolase AALDOA 15 Q08495 Dematin DMTN 15 P23276 Kell blood group glycoprotein KEL15 P69892 Hemoglobin subunit gamma-2 HBG2 14 P60174 Triosephosphateisomerase TPI1 14 P62258 14-3-3 protein epsilon YWHAE 14 O75326Semaphorin-7A SEMA7A 14 O75340 Programmed cell death protein 6 PDCD6 13E7EV99 Alpha-adducin ADD1 13 Q5VZU9 Tripeptidyl-peptidase 2 TPP2 13P53396 ATP-citrate synthase ACLY 13 C9J0K6 Sorcin SRI 12 P04406Glyceraldehyde-3-phosphate dehydrogenase GAPDH 12 P07738Bisphosphoglycerate mutase BPGM 12 B7Z3I9 Delta-aminolevulinic aciddehydratase ALAD 12 Q86X55 Histone-arginine methyltransferase CARM1CARM1 12 P07195 L-lactate dehydrogenase B chain; L-lactate dehydrogenaseLDHB 12 P23526 Adenosylhomocysteinase AHCY 12 Q32Q12 Nucleosidediphosphate kinase NME1-NME2 12 B7Z7A9 Phosphoglycerate kinase 1 PGK1 11P11166 Solute carrier family 2, facilitated glucose transporter member 1SLC2A1 11 P22303 Acetylcholinesterase ACHE 11 Q9NP58 ATP-bindingcassette sub-family B member 6, mitochondrial ABCB6 11 P40925 Malatedehydrogenase, cytoplasmic; Malate dehydrogenase MDH1 11 P00352 Retinaldehydrogenase 1 ALDH1A1 11 F2Z2V0 Copine-1 CPNE1 11 K7EMC9 WWdomain-binding protein 2 WBP2 10 F5H7S3 Tropomyosin alpha-1 chain TPM110 A6NN80 Annexin A6; Annexin ANXA6 10 P30043 Flavin reductase (NADPH)BLVRB 9 H7BXD5 Grancalcin GCA 9 P04083 Annexin A1; Annexin ANXA1 9P62937 Peptidyl-prolyl cis-trans isomerase PPIA 9 P37837 TransaldolaseTALDO1 9 Q06830 Peroxiredoxin-1 PRDX1 9 O75131 Copine-3 CPNE3 9 P00390Glutathione reductase, mitochondrial GSR 9 E7EQB2 Lactotransferrin LTF 9P07384 Calpain-1 catalytic subunit CAPN1 9 P02042 Hemoglobin subunitdelta HBD 8 P30086 Phosphatidylethanolamine-binding protein 1;Hippocampal cholinergic neurostimulating PEBP1 8 peptide P35613 BasiginBSG 8 Q9H0U4 Ras-related protein Rab-1B; Putative Ras-related proteinRab-1C RAB1B; RAB1C 8 P63092 Guanine nucleotide-binding protein G(s)subunit alpha isoforms GNAS 8 P48506 Glutamate-cysteine ligase catalyticsubunit GCLC 8 P06702 Protein S100-A9 S100A9 7 Q9UBV8 Peflin PEF1 7P17931 Galectin-3; Galectin LGALS3 7 P28066 Proteasome subunit alphatype-5 PSMA5 7 P07451 Carbonic anhydrase 3 CA3 7 E7EQ12 Calpastatin CAST7 P50895 Basal cell adhesion molecule BCAM 7 P28074 Proteasome subunitbeta type-5 PSMB5 7 G3V5Z7 Proteasome subunit alpha type; Proteasomesubunit alpha type-6 PSMA6 7 P25786 Proteasome subunit alpha type-1PSMA1 7 G3V1D3 Dipeptidyl peptidase 3 DPP3 7 P49247 Ribose-5-phosphateisomerase RPIA 7 Q5T9B7 Adenylate kinase isoenzyme 1 AK1 7 P25789Proteasome subunit alpha type-4; Proteasome subunit beta type PSMA4 7B4E022 Transketolase TKT 7 J3QS39 Ubiquitin-60S ribosomal protein L40;Ubiquitin; 60S ribosomal protein L40; Ubiquitin-40S UBB; RPS27A; 6ribosomal protein S27a; Ubiquitin; 40S ribosomal protein S27a;Polyubiquitin- UBC; UBA52; B; Ubiquitin; Polyubiquitin-C; UbiquitinUBBP4 H0Y7A7 Calmodulin CALM2 6 P28070 Proteasome subunit beta type-4PSMB4 6 Q9H4G4 Golgi-associated plant pathogenesis-related protein 1GLIPR2 6 Q9BY43 Charged multivesicular body protein 4a CHMP4A 6 P48426Phosphatidylinositol 5-phosphate 4-kinase type-2 alpha PIP4K2A 6 P28289Tropomodulin-1 TMOD1 6 P07911 Uromodulin; Uromodulin, secreted form UMOD6 Q9GZP4 PITH domain-containing protein 1 PITHD1 6 P78417 GlutathioneS-transferase omega-1 GSTO1 6 P25788 Proteasome subunit alpha type-3PSMA3 6 O14818 Proteasome subunit alpha type-7; Proteasome subunit alphatype-7-like PSMA7; PSMA8 6 P08107 Heat shock 70 kDa protein 1A/1B HSPA1A6 H0YD13 CD44 antigen CD44 6 P61225 Ras-related protein Rap-2b;Ras-related protein Rap-2c; Ras-related protein Rap-2a RAP2B; RAP2A; 6RAP2C P05109 Protein S100-A8; Protein S100-A8, N-terminally processedS100A8 6 P23528 Cofilin-1 CFL1 6 Q99808 Equilibrative nucleosidetransporter 1 SLC29A1 6 P84077 ADP-ribosylation factor 1;ADP-ribosylation factor 3; ADP-ribosylation factor 5; ADP- ARF1; ARF3; 6ribosylation factor 4 ARF5; ARF4 P31946 14-3-3 protein beta/alpha;14-3-3 protein beta/alpha, N-terminally processed YWHAB 6 C9JIS1 Guaninenucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2; Guaninenucleotide- GNB2; GNB4 6 binding protein subunit beta-4 P53990 IST1homolog IST1 6 Q99497 Protein DJ-1 PARK7 6 F5H7U0 6-phosphogluconatedehydrogenase, decarboxylating PGD 6 B7Z7E9 Aspartate aminotransferase,cytoplasmic GOT1 6 P62834 Ras-related protein Rap-1A; Ras-relatedprotein Rap-1b; Ras-related protein Rap-1b-like RAP1A; RAP1B 6 proteinP04899 Guanine nucleotide-binding protein G(i) subunit alpha-2 GNAI2 6P25325 3-mercaptopyruvate sulfurtransferase; Sulfurtransferase MPST 6Q9NP79 Vacuolar protein sorting-associated protein VTA1 homolog VTA1 6P00492 Hypoxanthine-guanine phosphoribosyltransferase HPRT1 6 Q16531 DNAdamage-binding protein 1 DDB1 6 P22314 Ubiquitin-likemodifier-activating enzyme 1 UBA1 6 P00441 Superoxide dismutase [Cu—Zn]SOD1 5 H7BY58 Protein-L-isoaspartate O-methyltransferase;Protein-L-isoaspartate(D-aspartate) O- PCMT1 5 methyltransferase P10768S-formylglutathione hydrolase ESD 5 P09543 2,3-cyclic-nucleotide3-phosphodiesterase CNP 5 P06733 Alpha-enolase; Enolase ENO1 5 P26038Moesin MSN 5 O75368 SH3 domain-binding glutamic acid-rich-like proteinSH3BGRL 5 K7EQ48 Glucose-6-phosphate isomerase GPI 5 P25787 Proteasomesubunit alpha type-2 PSMA2 5 P49721 Proteasome subunit beta type-2 PSMB25 F5H8J2 Protein disulfide-isomerase P4HB 5 C9J9P4 Phospholipidscramblase 1 PLSCR1 5 P09211 Glutathione S-transferase P GSTP1 5 B5MDF5GTP-binding nuclear protein Ran RAN 5 P07900 Heat shock protein HSP90-alpha HSP90AA1 5 K7EQ02 DAZ-associated protein 1 DAZAP1 5 Q13630GDP-L-fucose synthase TSTA3 5 F5H0T1 Stress-induced-phosphoprotein 1STIP1 5 P50502 Hsc70-interacting protein; Putative protein FAM10A5;Putative protein FAM10A4 ST13; ST13P5; 5 ST13P4 P20618 Proteasomesubunit beta type-1 PSMB1 5 P62805 Histone H4 HIST1H4A 5 P51148Ras-related protein Rab-5C RAB5C 5 H7C2G2 Ecto-ADP-ribosyltransferase 4ART4 5 J3KQ18 D-dopachrome decarboxylase; D-dopachromedecarboxylase-like protein DDT; DDTL 5 H3BPK3 Hydroxyacylglutathionehydrolase, mitochondrial HAGH 5 B4DIT7 Protein-glutaminegamma-glutamyltransferase 2 TGM2 5 O43633 Charged multivesicular bodyprotein 2a CHMP2A 5 B4DQH4 T-complex protein 1 subunit theta CCT8 5Q9UN37 Vacuolar protein sorting-associated protein 4A VPS4A 5 I3L397Eukaryotic translation initiation factor 5A-1; Eukaryotic translationinitiation factor 5A-1-like EIF5A; EIF5AL1 5 Q9Y5Z4 Heme-binding protein2 HEBP2 5 Q9UKV8 Protein argonaute-2 AGO2 5 F5H442 Tumor susceptibilitygene 101 protein TSG101 5 H3BLV0 Complement decay-accelerating factorCD55 5 P02008 Hemoglobin subunit zeta HBZ 5 E7EPV7 Alpha-synuclein SNCA4 P63104 14-3-3 protein zeta/delta YWHAZ 4 P02724 Glycophorin-A GYPA;GPErik 4 U3KQE2 Calpain small subunit 1 CAPNS1 4 Q9NP59 Solute carrierfamily 40 member 1 SLC40A1 4 Q5QPM9 Proteasome inhibitor PI31 subunitPSMF1 4 C9J8U2 Nicotinate phosphoribosyltransferase NAPRT 4 P6198114-3-3 protein gamma; 14-3-3 protein gamma, N-terminally processed YWHAG4 Q9UQ80 Proliferation-associated protein 2G4 PA2G4 4 E5RJR5 S-phasekinase-associated protein 1 SKP1 4 Q9NRV9 Heme-binding protein 1 HEBP1 4Q9H444 Charged multivesicular body protein 4b CHMP4B 4 P10599Thioredoxin TXN 4 Q5VSJ9 Blood group Rh(CE) polypeptide; Blood groupRh(D) polypeptide RHCE; RHD 4 Q9UK41 Vacuolar protein sorting-associatedprotein 28 homolog VPS28 4 Q9Y3I1 F-box only protein 7 FBXO7 4 P61026Ras-related protein Rab-10 RAB10 4 Q14974 Importin subunit beta-1 KPNB14 P27797 Calreticulin CALR 4 P18669 Phosphoglycerate mutase 1; Probablephosphoglycerate mutase 4 PGAM1; 4 PGAM4 P54725 UV excision repairprotein RAD23 homolog A RAD23A 4 P30613 Pyruvate kinase PKLR PKLR 4P63000 Ras-related C3 botulinum toxin substrate 1; Ras-related C3botulinum toxin substrate 3; Ras- RAC1; RAC3; 4 related C3 botulinumtoxin substrate 2 RAC2 P05164 Myeloperoxidase MPO 4 F5GWY2 Bifunctionalpurine biosynthesis protein PURH;Phosphoribosylaminoimidazolecarboxamide ATIC 4 formyltransferase; IMPcyclohydrolase P48637 Glutathione synthetase GSS 4 F5H5V4 26S proteasomenon-ATPase regulatory subunit 9 PSMD9 4 G5E9R5 Low molecular weightphosphotyrosine protein phosphatase ACP1 3 P46976 Glycogenin-1 GYG1 3P28072 Proteasome subunit beta type-6 PSMB6 3 P26447 Protein S100-A4S100A4 3 F5GXQ0 BRO1 domain-containing protein BROX BROX 3 P08754Guanine nucleotide-binding protein G(k) subunit alpha GNAI3 3 Q99436Proteasome subunit beta type-7 PSMB7 3 P62942 Peptidyl-prolyl cis-transisomerase FKBP1A 3 U3KQK0 Histone H2B HIST1H2B 3 J3QKR3 Proteasomesubunit beta type-3 PSMB3 3 P01116 GTPase KRas; GTPase KRas,N-terminally processed; GTPase HRas; GTPase HRas, N- KRAS; HRAS; 3terminally processed; GTPase NRas NRAS P13489 Ribonuclease inhibitorRNH1 3 Q08722 Leukocyte surface antigen CD47 CD47 3 Q5T123 SH3domain-binding glutamic acid-rich-like protein 3 SH3BGRL3 3 Q8WYQ7Galectin; Galectin-9 LGALS9 3 O75695 Protein XRP2 RP2 3 P00167Cytochrome b5 CYB5A 3 Q9Y4D1 Disheveled-associated activator ofmorphogenesis 1 DAAM1 3 P11021 78 kDa glucose-regulated protein HSPA5 3H7C1D4 Translin TSN 3 P07737 Profilin-1 PFN1 3 M0R389Platelet-activating factor acetylhydrolase IB subunit gamma PAFAH1B3 3A6NJA2 Ubiquitin carboxyl-terminal hydrolase 14 USP14 3 P10644cAMP-dependent protein kinase type l-alpha regulatory subunit PRKAR1A 3Q9BS40 Latexin LXN 3 G5EA52 Protein disulfide-isomerase A3 PDIA3 3P53004 Biliverdin reductase A BLVRA 3 Q04656 Copper-transporting ATPase1 ATP7A 3 H9KV70 Neutrophil gelatinase-associated lipocalin LCN2 3O00299 Chloride intracellular channel protein 1 CLIC1 3 F8WF69 Clathrinlight chain A CLTA 3 G3V2F7 Ubiquitin-conjugating enzyme E2 variant 1;Ubiquitin-conjugating enzyme E2 variant 2 UBE2V1; 3 UBE2V2 F8WDS9LanC-like protein 1 LANCL1 3 P60891 Ribose-phosphate pyrophosphokinase 1PRPS1 3 K7ESE8 Bleomycin hydrolase BLMH 3 H0YNE3 Proteasome activatorcomplex subunit 1 PSME1 3 P16930 Fumarylacetoacetase FAH 3 F8VSD4Ubiquitin-conjugating enzyme E2 N UBE2N 3 P07203 Glutathione peroxidase1 GPX1 3 P62328 Thymosin beta-4; Hematopoietic system regulatory peptideTMSB4X; 3 TMSB4XP4 E5RIW3 Tubulin-specific chaperone A TBCA 3 M0R0Y2Alpha-soluble NSF attachment protein NAPA 3 P15374 Ubiquitincarboxyl-terminal hydrolase isozyme L3 UCHL3 3 P04921 Glycophorin-C GYPC2 H0YDI1 Lymphocyte function-associated antigen 3 CD58 2 B4E220Aquaporin-1 AQP1 2 C9JEN3 Protein lifeguard 3 TMBIM1 2 F5H2R5 RhoGDP-dissociation inhibitor 2 ARHGDIB 2 Q53TN4 Cytochrome b reductase 1CYBRD1 2 Q9NZD4 Alpha-hemoglobin-stabilizing protein AHSP 2 Q8NHG7 SmallVCP/p97-interacting protein SVIP 2 Q5JYX0 Cell division control protein42 homolog CDC42 2 Q71RC9 Small integral membrane protein 5 SMIM5 2E9PNW4 CD59 glycoprotein CD59 2 P09105 Hemoglobin subunit theta-1 HBQ1 2R4GN98 Protein S100; Protein S100-A6 S100A6 2 O75531Barrier-to-autointegration factor; Barrier-to-autointegration factor,N-terminally processed BANF1 2 Q5T6W5 Heterogeneous nuclearribonucleoprotein K HNRNPK 2 F5H4Q5 Vacuolar protein sorting-associatedprotein 37C VPS37C 2 J3QK90 NSFL1 cofactor p47 NSFL1C 2 H3BV85 BolA-likeprotein 2 BOLA2B; BOLA2 2 Q9NRX4 14 kDa phosphohistidine phosphatasePHPT1 2 H3BS66 Small integral membrane protein 1 SMIM1 2 E7ESC6Exportin-7 XPO7 2 P68402 Platelet-activating factor acetyl hydrolase IBsubunit beta PAFAH1B2 2 Q9BRF8 Serine/threonine-protein phosphataseCPPED1 CPPED1 2 P08246 Neutrophil elastase ELANE 2 E9PN50 26S proteaseregulatory subunit 6A PSMC3 2 E7EUC7 UTP-glucose-1-phosphateuridylyltransferase UGP2 2 B8ZZB8 CB1 cannabinoid receptor-interactingprotein 1 CNRIP1 2 E9PCS3 26S proteasome non-ATPase regulatory subunit 2PSMD2 2 P59666 Neutrophil defensin 3; HP 3-56; Neutrophil defensin 2;Neutrophil defensin 1; HP 1- DEFA3; DEFA1 2 56; Neutrophil defensin 2O15400 Syntaxin-7 STX7 2 P00338 L-lactate dehydrogenase A chain LDHA 2P61970 Nuclear transport factor 2 NUTF2 2 E7EMV0 Protein diaphanoushomolog 1 DIAPH1 2 F5GY90 Porphobilinogen deaminase HMBS 2 P61020Ras-related protein Rab-5B RAB5B 2 Q99828 Calcium and integrin-bindingprotein 1 CIB1 2 B4DUA0 Plastin-2 LCP1 2 C9JTY3 Protein TFG TFG 2 P2734814-3-3 protein theta YWHAG 2 H0YKZ7 Annexin; Annexin A2; Putativeannexin A2-like protein ANXA2; 2 ANXA2P2 P08238 Heat shock protein HSP90-beta HSP90AB1 2 J3KQP6 Ras-related protein Rab-11B; Ras-relatedprotein Rab-11A RAB11A; 2 RAB11B A6NMU3 Signal transducing adaptermolecule 1 STAM 2 P53985 Monocarboxylate transporter 1 SLC16A1 2 F6USW4F-actin-capping protein subunit beta CAPZB 2 O14964 Hepatocyte growthfactor-regulated tyrosine kinase substrate HGS 2 P20020 Plasma membranecalcium-transporting ATPase 1; Calcium-transporting ATPase ATP2B1 2P36959 GMP reductase 1 GMPR 2 Q9Y376 Calcium-binding protein 39 CAB39 2Q9Y6M5 Zinc transporter 1 SLC30A1 2 Q8IZ83 Aldehyde dehydrogenase family16 member A1 ALDH16A1 2 Q99459 Cell division cycle 5-like protein CDC5L2 P06132 Uroporphyrinogen decarboxylase UROD 2 J3KNT0 Fascin FSCN1 2P49189 4-trimethylaminobutyraldehyde dehydrogenase ALDH9A1 2 H3BNT7 26Sproteasome non-ATPase regulatory subunit 7 PSMD7 2 P05023Sodium/potassium-transporting ATPase subunit alpha-1;Sodium/potassium-transporting ATP1A1; 2 ATPase subunit alpha-3;Sodium/potassium-transporting ATPase subunit alpha- ATP1A2; 2;Sodium/potassium-transporting ATPase subunit alpha-4;Potassium-transporting ATPase ATP1A3; alpha chain 1;Potassium-transporting ATPase alpha chain 2 ATP1A4; ATP4A; ATP12A P34932Heat shock 70 kDa protein 4 HSPA4 2 K7EMV3 Histone H3 H3F3B 2 Q8IU68Transmembrane channel-like protein 8 TMC8 2 E7ENZ3 T-complex protein 1subunit epsilon CCT5 2 Q5TZA2 Rootletin CROCC 2 Q9P203 BTB/POZdomain-containing protein 7 BTBD7 2 Q7LBR1 Charged multivesicular bodyprotein 1b CHMP1B 2 U3KQ56 Glyoxylate reductase/hydroxypyruvatereductase GRHPR 2 H0YJ11 Alpha-actinin-1; Alpha-actinin-2;Alpha-actinin-4 ACTN1; ACTN4; 2 ACTN2 Q9UDT6 CAP-Gly domain-containinglinker protein 2 CLIP2 2 P09960 Leukotriene A-4 hydrolase LTA4H 2 Q5HY54Filamin-A FLNA 2 E9PJL5 Uncharacterized protein C12orf55; Putativeuncharacterized protein C12orf63 C12orf55; 2 C12orf63 G3V2U7Acylphosphatase; Acylphosphatase-1 FBN3; ACYP1 2 E9PQN4 Complementreceptor type 1 CR1 2 Q9BSL1 Ubiquitin-associated domain-containingprotein 1 UBAC1 2 Q04917 14-3-3 protein eta YWHAH 2 B7ZBP9Serine/threonine-protein phosphatase 2A activator PPP2R4; DKF 2Zp781M17165 Q8NDC0 MAPK-interacting and spindle-stabilizing protein-likeMAPK1IP1L 1 P68133 Actin, alpha skeletal muscle; Actin, alpha cardiacmuscle 1; Actin, gamma-enteric smooth ACTA1; ACTC1; 1 muscle; Actin,aortic smooth muscle ACTG2; ACTA2 P69891 Hemoglobin subunit gamma-1 HBG11 S4R3Y4 Protein AM BP; Alpha-1-microglobulin; Inter-alpha-trypsininhibitor light chain; Trypstatin AMBP 1 I3L3E4 Charged multivesicularbody protein 6 CHMP6 1 Q16570 Atypical chemokine receptor 1 ACKR1 1Q5VY30 Retinol-binding protein 4; Plasma retinol-binding protein(1-182);Plasma retinol-binding RBP4 1 protein(1-181); Plasma retinol-bindingprotein(1-179); Plasma retinol-binding protein(1-176) E7END7 Ras-relatedprotein Rab-1A RAB1A 1 Q5VU59 TPM3 1 P17066 Heat shock 70 kDa protein 6;Putative heat shock 70 kDa protein 7 HSPA6; HSPA7 1 Q04760Lactoylglutathione lyase GLO1 1 D6RD66 WD repeat-containing protein 1WDR1 1 K7EM02 Katanin p60 ATPase-containing subunit A-like 2 KATNAL2 1P14209 CD99 antigen CD99 1 E9PIR7 Thioredoxin reductase 1, cytoplasmicGML; TXNRD1 1 K7EMQ9 EIF3K 1 P15531 Nucleoside diphosphate kinase A NME11 H7BZT4 Small ubiquitin-related modifier 4; Small ubiquitin-relatedmodifier 2; Small ubiquitin-related SUMO2; SUMO3; 1 modifier 3 SUMO4O00560 Syntenin-1 SDCBP 1 Q9BVM4 Gamma-glutamylaminecyclotransferaseGGACT 1 K7EKH5 Fructose-bisphosphate aldolase C ALDOC 1 P49773 Histidinetriad nucleotide-binding protein 1 HINT1 1 H0YBY6 Disks large-associatedprotein 2 DLGAP2 1 Q9Y624 Junctional adhesion molecule A F11R 1 B1AKQ8Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1;Guanine nucleotide- GNB1; GNB3 1 binding protein G(I)/G(S)/G(T) subunitbeta-3 K7EKN6 Urea transporter 1 SLC14A1 1 I3L0K2 Thioredoxindomain-containing protein 17 TXNDC17 1 A8MXY0 Syntaxin-4 STX4 1 O14773Tripeptidyl-peptidase 1 TPP1 1 E9PNW0 Nucleosome assembly protein 1-like1; Nucleosome assembly protein 1-like 4 NAP1L4; 1 NAP1L1 Q5TDH0 ProteinDDI1 homolog 2 DDI2 1 Q96JM4 Leucine-rich repeat and IQdomain-containing protein 1 LRRIQ1 1 F5GWT9Phosphoribosylformylglycinamidine synthase PFAS 1 F2Z3J2 26S proteasomenon-ATPase regulatory subunit 5 PSMD5 1 J3QL74 Zinc finger and BTBdomain-containing protein 14 ZBTB14 1 E9PJC7 CD82 antigen CD82 1 Q9H936Mitochondrial glutamate carrier 1 SLC25A22 1 D6RD63 COP9 signalosomecomplex subunit 4 COPS4 1 Q6B0K9 Hemoglobin subunit mu HBM 1 Q31611 HLAclass I histocompatibility antigen, alpha chain G HLA-G 1 H7BY04 Lamininsubunit gamma-3 LAMC3 1 Q9UL25 Ras-related protein Rab-21 RAB21 1 H7C3P7Ras-related protein Ral-A RALA 1 P08311 Cathepsin G CTSG 1 E9PE37Ras-related protein Rab-2A; Ras-related protein Rab-2B RAB2B; RAB2A 1G3V1N2 HBA2 1 P00387 NADH-cytochrome b5 reductase 3; NADH-cytochrome b5reductase 3 membrane-bound CYB5R3 1 form; NADH-cytochrome b5 reductase 3soluble form O75339 Cartilage intermediate layer protein 1; Cartilageintermediate layer protein 1 C1; Cartilage CILP 1 intermediate layerprotein 1 C2 P14324 Farnesyl pyrophosphate synthase FDPS 1 K7EKG2Thioredoxin-like protein 1 TXNL1 1 (B). UNIPROT COMPLETE PROTEOME HOMOSAPIENS DATABASE WHEN THE HEMOGLOBIN WAS ANALYZED SEPARATELY P02549Spectrin alpha chain, erythrocytic 1 SPTA1 197 P11277 Spectrin betachain, erythrocytic SPTB 177 P16157 Ankyrin-1 ANK1 94 P55072Transitional endoplasmic reticulum ATPase VCP 53 P111714 Protein 4.1EPB41 52 P35579 Myosin-9 MYH9 44 Q8WUM4 Programmed cell death6-interacting protein PDCD6IP 43 P02730 Band 3 anion transport proteinSLC4A1 43 P16452 Erythrocyte membrane protein band 4.2 EPB42 42 P04040Catalase CAT 40 A0A087WVQ6 Clathrin heavy chain; Clathrin heavy chain 1CLTC 38 P35612 Beta-adducin ADD2 37 P16157 Ankyrin-1 ANK1 36 Q14254Flotillin-2 FLOT2 33 P20073 Annexin A7 ANXA7 31 O75955 Flotillin-1 FLOT130 P53396 ATP-citrate synthase ACLY 30 P06753 TPM3 29 P49368 T-complexprotein 1 subunit gamma CCT3 29 P236344 Plasma membranecalcium-transporting ATPase 4 ATP2B4 28 P11142 Heat shock cognate 71 kDaprotein HSPA8 28 P60709 Actin, cytoplasmic 1; Actin, cytoplasmic 1,N-terminally processed ACTB 28 Q5T4S7 E3 ubiquitin-protein ligase UBR4UBR4 27 P78371 T-complex protein 1 subunit beta CCT2 26 P28289Tropomodulin-1 TMOD1 26 P50395 Rab GDP dissociation inhibitor beta GDI225 P27105 Erythrocyte band 7 integral membrane protein STOM 25 P68871Hemoglobin subunit beta; LW-hemorphin-7; Spinorphin HBB 25 P02730 Band 3anion transport protein SLC4A1 25 Q00013 55 kDa erythrocyte membraneprotein MPP1 24 J3KPS3 Fructose-bisphosphate aldolase;Fructose-bisphosphate aldolase A ALDOA 24 P00352 Retinal dehydrogenase 1ALDH1A1 24 Q86VP6 Cullin-associated NEDD8-dissociated protein 1 CAND1 24P49327 Fatty acid synthase FASN 24 Q13228 Selenium-binding protein 1SELENBP1 22 P00915 Carbonic anhydrase 1 CA1 22 P50991 T-complex protein1 subunit delta CCT4 22 P50990 T-complex protein 1 subunit theta CCT8 22P32119 Peroxiredoxin-2 PRDX2 21 P50995 Annexin A11 ANXA11 21 P69905Hemoglobin subunit alpha HBA1 21 P00558 Phosphoglycerate kinase 1 PGK120 H7BXK9 ATP-binding cassette sub-family B member 6, mitochondrialABCB6 20 P08758 Annexin A5; Annexin ANXA5 20 P09525 Annexin A4; AnnexinANXA4 20 P07900 Heat shock protein HSP 90-alpha HSP90AA1 20 P48643T-complex protein 1 subunit epsilon CCT5 20 P07384 Calpain-1 catalyticsubunit CAPN1 20 P22314 Ubiquitin-like modifier-activating enzyme 1 UBA119 P04406 Glyceraldehyde-3-phosphate dehydrogenase GAPDH 19 Q08495Dematin DMTN 19 Q99832 T-complex protein 1 subunit eta CCT7 19 P29144Tripeptidyl-peptidase 2 TPP2 19 P30041 Peroxiredoxin-6 PRDX6 18 E9PM6926S protease regulatory subunit 6A PSMC3 18 P40227 T-complex protein 1subunit zeta CCT6A 18 P50570 Dynamin-2 DNM2 18 E7EQB2 Lactotransferrin;Lactoferricin-H; Kaliocin-1; Lactoferroxin-A; Lactoferroxin-B;Lactoferroxin-C LTF 18 P31948 Stress-induced-phosphoprotein 1 STIP1 18E7ESC6 Exportin-7 XPO7 18 E7EV99 Alpha-adducin ADD1 17 P62258 14-3-3protein epsilon YWHAE 17 P30613 Pyruvate kinase PKLR PKLR 17 A0A0G2JIW1Heat shock 70 kDa protein 1B; Heat shock 70 kDa protein 1A HSPA1B; 17HSPA1A Q16531 DNA damage-binding protein 1 DDB1 17 P11021 78 kDaglucose-regulated protein HSPA5 17 F5H2F4 C-1-tetrahydrofolate synthase,cytoplasmic; Methylenetetrahydrofolate dehydrogenase MTHFD1 17 P07195L-lactate dehydrogenase B chain; L-lactate dehydrogenase LDHB 16 P45974Ubiquitin carboxyl-terminal hydrolase 5 USP5 16 O43242 26S proteasomenon-ATPase regulatory subunit 3 PSMD3 16 C9J0K6 Sorcin SRI 16 P08133Annexin A6; Annexin ANXA6 16 Q13200 26S proteasome non-ATPase regulatorysubunit 2 PSMD2 16 P23276 Kell blood group glycoprotein KEL 16 P34932Heat shock 70 kDa protein 4 HSPA4 16 A0A0A0MSI0 Peroxiredoxin-1 PRDX1 16Q9Y230 RuvB-like 2 RUVBL2 16 Q5XPI4 E3 ubiquitin-protein ligase RNF123RNF123 16 P68871 Hemoglobin subunit beta; LW-hemorphin-7; Spinorphin HBB15 P60174 Triosephosphate isomerase TPI1 15 P00491 Purine nucleosidephosphorylase PNP 15 C9JIF9 Acylamino-acid-releasing enzyme APEH 15H7BYY1 Tropomyosin alpha-1 chain TPM1 15 P35998 26S protease regulatorysubunit 7 PSMC2 15 P17987 T-complex protein 1 subunit alpha TCP1 15P09543 2,3-cyclic-nucleotide 3-phosphodiesterase CNP 15 Q99460 26Sproteasome non-ATPase regulatory subunit 1 PSMD1 15 Q9Y4E8 Ubiquitincarboxyl-terminal hydrolase 15 USP15 15 Q9C0C9 E2/E3 hybridubiquitin-protein ligase UBE2O UBE2O 15 P26038 Moesin MSN 15 P04083Annexin A1; Annexin ANXA1 14 P30043 Flavin reductase (NADPH) BLVRB 14P11166 Solute carrier family 2, facilitated glucose transporter member 1SLC2A1 14 P00918 Carbonic anhydrase 2 CA2 14 P06733 Alpha-enolase ENO114 Q5TDH0 Protein DDI1 homolog 2 DDI2 14 B0QZ18 Copine-1 CPNE1 14 O75326Semaphorin-7A SEMA7A 14 P05164 Myeloperoxidase MPO 14 Q9Y265 RuvB-like 1RUVBL1 14 P29401 Transketolase TKT 14 I3L0N3 Vesicle-fusing ATPase NSF14 Q4VB86 Protein 4.1 EPB41 14 P11277 Spectrin beta chain, erythrocyticSPTB 14 P13716 Delta-aminolevulinic acid dehydratase ALAD 13 P07738Bisphosphoglycerate mutase BPGM 13 P48506 Glutamate-cysteine ligasecatalytic subunit GCLC 13 Q99816 Tumor susceptibility gene 101 proteinTSG101 13 O14818 Proteasome subunit alpha type-7 PSMA7 13 P23526Adenosylhomocysteinase AHCY 13 P61225 Ras-related protein Rap-2b RAP2B13 O00231 26S proteasome non-ATPase regulatory subunit 11 PSMD11 13P11413 Glucose-6-phosphate 1-dehydrogenase G6PD 13 P00338 L-lactatedehydrogenase A chain LDHA 12 Q99808 Equilibrative nucleosidetransporter 1 SLC29A1 12 A6NJA2 Ubiquitin carboxyl-terminal hydrolase;Ubiquitin carboxyl-terminal hydrolase 14 USP14 12 Q06323 Proteasomeactivator complex subunit 1 PSME1 12 P28074 Proteasome subunit betatype-5 PSMB5 12 B3KQV6 Serine/threonine-protein phosphatase 2A 65 kDaregulatory subunit A alpha isoform PPP2R1A 12 Q14974 Importin subunitbeta-1 KPNB1 12 P25786 Proteasome subunit alpha type-1; Proteasomesubunit alpha type PSMA1 12 Q86X55 Histone-arginine methyltransferaseCARM1 CARM1 12 A6NG10 WW domain-binding protein 2 WBP2 12 P63092 Guaninenucleotide-binding protein G(s) subunit alpha GNAS 12 P31939Bifunctional purine biosynthesis protein PURH;Phosphoribosylaminoimidazolecarboxamide ATIC 12 formyltransferase; IMPcyclohydrolase P52209 6-phosphogluconate dehydrogenase, decarboxylatingPGD 12 A0A087X0C8 Calpain-5 CAPN5 12 F8W9S7 GTPase-activating proteinand VPS9 domain-containing protein 1 GAPVD1 12 P60842 Eukaryoticinitiation factor 4A-I EIF4A1 12 P69905 Hemoglobin subunit alpha HBA1 11Q6XQN6 Nicotinate phosphoribosyltransferase NAPRT 11 P48637 Glutathionesynthetase GSS 11 H7BZ94 Protein disulfide-isomerase P4HB 11 P21980Protein-glutamine gamma-glutamyltransferase 2 TGM2 11 P50895 Basal celladhesion molecule BCAM 11 A0A087X2I1 26S protease regulatory subunit 10BPSMC6 11 G3V1D3 Dipeptidyl peptidase 3 DPP3 11 P05023Sodium/potassium-transporting ATPase subunit alpha-1;Sodium/potassium-transporting ATP1A1; ATP1A3 11 ATPase subunit alpha-3P40925 Malate dehydrogenase, cytoplasmic; Malate dehydrogenase MDH1 11Q9UKV8 Protein argonaute-2 AGO2 11 P30566 Adenylosuccinate lyase ADSL 11P20618 Proteasome subunit beta type-1 PSMB1 11 P17858 ATP-dependent6-phosphofructokinase, liver type PFKL 11 A0A087X253 AP-2 complexsubunit beta AP2B1 11 O95782 AP-2 complex subunit alpha-1 AP2A1 11O00232 26S proteasome non-ATPase regulatory subunit 12 PSMD12 11 Q9BSL1Ubiquitin-associated domain-containing protein 1 UBAC1 11 A0A087WUL0Bifunctional ATP-dependent dihydroxyacetone kinase/FAD-AMP lyase(cyclizing); ATP- TKFC; DAK 11 dependent dihydroxyacetone kinase;FAD-AMP lyase (cyclizing) P69891 Hemoglobin subunit gamma-1 HBG1 11A0A087WZE4 Spectrin alpha chain, erythrocytic 1 SPTA1 11 P48426Phosphatidylinositol 5-phosphate 4-kinase type-2 alpha PIP4K2A 10 P10644cAMP-dependent protein kinase type I-alpha regulatory subunit PRKAR1A 10Q9BWD1 Acetyl-CoA acetyltransferase, cytosolic ACAT2 10 P62191 26Sprotease regulatory subunit 4 PSMC1 10 E9PBS1 Multifunctional proteinADE2; Phosphoribosylaminoimidazole-succinocarboxamide PAICS 10 synthase;Phosphoribosylaminoimidazole carboxylase M0R0Y2 Alpha-soluble NSFattachment protein NAPA 10 P78417 Glutathione S-transferase omega-1GSTO1 10 P25789 Proteasome subunit alpha type-4; Proteasome subunitalpha type; Proteasome subunit beta PSMA4 10 type Q9H0U4 Ras-relatedprotein Rab-1B; Putative Ras-related protein Rab-1C RAB1B; RAB1C 10Q16401 26S proteasome non-ATPase regulatory subunit 5 PSMD5 10 P30101Protein disulfide-isomerase A3 PDIA3 10 D6RAX7 COP9 signalosome complexsubunit 4 COPS4 10 O75340 Programmed cell death protein 6 PDCD6 10Q96P70 Importin-9 IPO9 10 P38606 V-type proton ATPase catalytic subunitA ATP6V1A 10 A0A0G2JH68 Protein diaphanous homolog 1 DIAPH1 10 Q5T9B7Adenylate kinase isoenzyme 1 AK1 10 O14980 Exportin-1 XPO1 10 P43686 26Sprotease regulatory subunit 6B PSMC4 10 H0YH81 ATP synthase subunitbeta; ATP synthase subunit beta, mitochondrial ATP5B 10 Q04656Copper-transporting ATPase 1 ATP7A 10 P16452 Erythrocyte membraneprotein band 4.2 EPB42 10 Q9BY43 Charged multivesicular body protein 4aCHMP4A 9 P51148 Ras-related protein Rab-5C RAB5C 9 Q9NRV9 Heme-bindingprotein 1 HEBP1 9 Q9UNZ2 NSFL1 cofactor p47 NSFL1C 9 Q16851UTP--glucose-1-phosphate uridylyltransferase UGP2 9 P28066 Proteasomesubunit alpha type-5 PSMA5 9 A0A0C4DGQ5 Calpain small subunit 1 CAPNS1 9A0A087X1Z3 Proteasome activator complex subunit 2 PSME2 9 Q01518Adenylyl cyclase-associated protein 1 CAP1 9 B1AKQ8 Guaninenucleotide-binding protein G(I)/G(S)/G(T) subunit beta-1 GNB1 9 O75131Copine-3 CPNE3 9 P54725 UV excision repair protein RAD23 homolog ARAD23A 9 P11215 lntegrin alpha-M ITGAM 9 Q93008 Probable ubiquitincarboxyl-terminal hydrolase FAF-X USP9X 9 Q96G03 Phosphoglucomutase-2PGM2 9 P49721 Proteasome subunit beta type-2 PSMB2 9 Q15008 26Sproteasome non-ATPase regulatory subunit 6 PSMD6 9 Q9UNQ0 ATP-bindingcassette sub-family G member 2 ABCG2 9 P22303 Acetylcholinesterase;Carboxylic ester hydrolase ACHE 9 G3V5Z7 Proteasome subunit alpha type;Proteasome subunit alpha type-6 PSMA6 9 O15439 Multidrugresistance-associated protein 4 ABCC4 9 P37837 Transaldolase TALDO1 9O14744 Protein arginine N-methyltransferase 5 PRMT5 9 P02042 Hemoglobinsubunit delta HBD 9 P25788 Proteasome subunit alpha type-3 PSMA3 8J3QS39 Ubiquitin-60S ribosomal protein L40; Ubiquitin; 60S ribosomalprotein L40; Ubiquitin-40S UBB; RPS27A; 8 ribosomal protein S27a;Ubiquitin; 40S ribosomal protein S27a; Polyubiquitin- UBC; UBA52; B;Ubiquitin; Polyubiquitin-C; Ubiquitin UBBP4 O94919 Endonucleasedomain-containing 1 protein ENDOD1 8 P31946 14-3-3 protein beta/alphaYWHAB 8 P60891 Ribose-phosphate pyrophosphokinase 1; Ribose-phosphatepyrophosphokinase 2; Ribose- PRPS1; PRPS2; 8 phosphate pyrophosphokinase3 PRPS1L1 P62195 26S protease regulatory subunit 8 PSMC5 8 A0A024RA52Proteasome subunit alpha type; Proteasome subunit alpha type-2 PSMA2 8P04899 Guanine nucleotide-binding protein G(i) subunit alpha-2 GNAI2 8Q13561 Dynactin subunit 2 DCTN2 8 P53004 Biliverdin reductase A BLVRA 8P00387 NADH-cytochrome b5 reductase 3 CYB5R3 8 P16152 Carbonyl reductase[NADPH] 1 CBR1 8 P06744 Glucose-6-phosphate isomerase GPI 8 Q99733Nucleosome assembly protein 1-like 4 NAP1L4 8 P04792 Heat shock proteinbeta-1 HSPB1 8 P17612 cAMP-dependent protein kinase catalytic subunitalpha; cAMP-dependent protein kinase PRKACA; KIN27; 8 catalytic subunitbeta PRKACB P20340 Ras-related protein Rab-6A RAB6A 8 P13796 Plastin-2LCP1 8 P52907 F-actin-capping protein subunit alpha-1 CAPZA1 8 Q14697Neutral alpha-glucosidase AB GANAB 8 P08514 Integrin alpha-IIb; Integrinalpha-IIb heavy chain; Integrin alpha-IIb light chain, form 1; IntegrinITGA2B 8 alpha-IIb light chain, form 2 P26641 Elongation factor 1-gammaEEF1G 8 Q9UQ80 Proliferation-associated protein 2G4 PA2G4 8 Q9Y4D1Disheveled-associated activator of morphogenesis 1 DAAM1 8 P11166 Solutecarrier family 2, facilitated glucose transporter member 1 SLC2A1 8P63261 Actin, cytoplasmic 2; Actin, cytoplasmic 2, N-terminallyprocessed; Actin, cytoplasmic 1; Actin, ACTG1; ACTB; 8 cytoplasmic 1,N-terminally processed; Actin, gamma-enteric smooth muscle; Actin, alphaACTG2; ACTA1; skeletal muscle; Actin, alpha cardiac muscle 1; Actin,aortic smooth muscle ACTC1; ACTA2 P02008 Hemoglobin subunit zeta HBZ 8P20073 Annexin A7 ANXA7 8 P35613 Basigin BSG 7 P49720 Proteasome subunitbeta type-3 PSMB3 7 P17931 Galectin-3; Galectin LGALS3 7 P63104 14-3-3protein zeta/delta YWHAZ 7 Q9H444 Charged multivesicular body protein 4bCHMP4B 7 O43396 Thioredoxin-like protein 1 TXNL1 7 P50502Hsc70-interacting protein; Putative protein FAM10A4; Putative proteinFAM10A5 ST13; ST13P4; 7 ST13P5 E7EQ12 Calpastatin CAST 7 P491894-trimethylaminobutyraldehyde dehydrogenase ALDH9A1 7 Q9Y3I1 F-box onlyprotein 7 FBXO7 7 P07954 Fumarate hydratase, mitochondrial FH 7 F6S8N6Protein-L-isoaspartate O-methyltransferase;Protein-L-isoaspartate(D-aspartate) O- PCMT1 7 methyltransferase P49247Ribose-5-phosphate isomerase RPIA 7 P62834 Ras-related protein Rap-1ARAP1A 7 A0A087WUQ6 Glutathione peroxidase; Glutathione peroxidase 1 GPX17 O60256 Phosphoribosyl pyrophosphate synthase-associated protein 2PRPSAP2 7 Q00796 Sorbitol dehydrogenase SORD 7 O00299 Chlorideintracellular channel protein 1 CLIC1 7 Q99497 Protein deglycase DJ-1PARK7 7 P17174 Aspartate aminotransferase, cytoplasmic GOT1 7 H7BXD5Grancalcin GCA 7 E9PGT1 Translin TSN 7 C9J7K9 Phospholipid scramblase 1PLSCR1 7 Q13618 Cullin-3 CUL3 7 O75695 Protein XRP2 RP2 7 P09960Leukotriene A-4 hydrolase LTA4H 7 E9PLK3 Puromycin-sensitiveaminopeptidase NPEPPS 7 P00492 Hypoxanthine-guaninephosphoribosyltransferase HPRT1 7 F5H4B6 Aldehyde dehydrogenase family16 member A1 ALDH16A1 7 D6RA82 Annexin; Annexin A3 ANXA3 7 P61106Ras-related protein Rab-14 RAB14 7 Q16775 Hydroxyacylglutathionehydrolase, mitochondrial HAGH 7 P07355 Annexin A2; Annexin; Putativeannexin A2-like protein ANXA2; ANXA2P2 7 A0A087WX08 Gamma-adducin ADD3 7P08238 Heat shock protein HSP 90-beta HSP90AB1 7 Q8IZY2 ATP-bindingcassette sub-family A member 7 ABCA7 7 P14780 Matrix metalloproteinase-9MMP9 7 P12955 Xaa-Pro dipeptidase PEPD 7 P68371 Tubulin beta-4B chain;Tubulin beta-4A chain; Tubulin beta chain TUBB4B; 7 TUBB4A; TUBB O15067Phosphoribosylformylglycinamidine synthase PFAS 7 P35241 Radixin RDX 7O60488 Long-chain-fatty-acid--CoA ligase 4 ACSL4 7 A0A0C4DGX4 Cullin-1CUL1 7 P50148 Guanine nucleotide-binding protein G(q) subunit alpha GNAQ7 Q15907 Ras-related protein Rab-11B; Ras-related protein Rab-11ARAB11B; 7 RAB11A P30086 Phosphatidylethanolamine-binding protein 1;Hippocampal cholinergic neurostimulating PEBP1 6 peptide P55036 26Sproteasome non-ATPase regulatory subunit 4 PSMD4 6 P28070 Proteasomesubunit beta type-4 PSMB4 6 Q9UNS2 COP9 signalosome complex subunit 3COPS3 6 P08754 Guanine nucleotide-binding protein G(k) subunit alphaGNAI3 6 P61006 Ras-related protein Rab-8A RAB8A 6 P61019 Ras-relatedprotein Rab-2A RAB2A 6 H0Y8C6 Importin-5 IPO5 6 Q00577 Transcriptionalactivator protein Pur-alpha PURA 6 P52565 Rho GDP-dissociation inhibitor1 ARHGDIA 6 Q9Y5Z4 Heme-binding protein 2 HEBP2 6 J3KNF4 Copperchaperone for superoxide dismutase; Superoxide dismutase [Cu—Zn] CCS 6A0A087WXS7 ATPase ASNA1 ASNA1 6 P05089 Arginase-1 ARG1 6 O953366-phosphogluconolactonase PGLS 6 Q92508 Piezo-type mechanosensitive ionchannel component 1 PIEZO1 6 P84077 ADP-ribosylation factor 1;ADP-ribosylation factor 3 ARF1; ARF3 6 P21281 V-type proton ATPasesubunit B, brain isoform; V-type proton ATPase subunit B, kidneyATP6V1B2; 6 isoform ATP6V1B1 P04259 Keratin, type II cytoskeletal 6BKRT6B 6 Q92905 COP9 signalosome complex subunit 5 COPS5 6 P61163Alpha-centractin ACTR1A 6 O95373 Importin-7 IPO7 6 C9JD73 Proteinphosphatase 1 regulatory subunit 7 PPP1R7 6 Q99536 Synaptic vesiclemembrane protein VAT-1 homolog VAT1 6 Q86UX7 Fermitin family homolog 3FERMT3 6 C9JFE4 COP9 signalosome complex subunit 1 GPS1 6 P36959 GMPreductase 1 GMPR 6 B5MDF5 GTP-binding nuclear protein Ran RAN 6 F5GY90Porphobilinogen deaminase HMBS 6 E7EX90 Dynactin subunit 1 DCTN1 6H0Y512 Adipocyte plasma membrane-associated protein APMAP 6 Q32Q12Nucleoside diphosphate kinase; Nucleoside diphosphate kinase B; Putativenucleoside NME1-NME2; 6 diphosphate kinase NME2; NME1; NME2P1 P13807Glycogen [starch] synthase, muscle GYS1 6 K7ES02 Bleomycin hydrolaseBLMH 6 P17213 Bactericidal permeability-increasing protein BPI 6 Q9UNM626S proteasome non-ATPase regulatory subunit 13 PSMD13 6 J3KQ32 Obg-likeATPase 1 OLA1 6 Q9Y490 Talin-1 TLN1 6 H0YD13 CD44 antigen CD44 6 P18669Phosphoglycerate mutase 1; Phosphoglycerate mutase 2; Probablephosphoglycerate PGAM1; PGAM2; 6 mutase 4 PGAM4 P23528 Cofilin-1 CFL1 6Q5SR44 Complement receptor type 1 CR1 6 Q99436 Proteasome subunit betatype-7 PSMB7 6 P47756 F-actin-capping protein subunit beta CAPZB 6P30740 Leukocyte elastase inhibitor SERPINB1 6 A0A024R571 EHdomain-containing protein 1 EHD1 6 P30043 Flavin reductase (NADPH) BLVRB6 P32119 Peroxiredoxin-2 PRDX2 6 C9J0K6 Sorcin SRI 6 P27105 Erythrocyteband 7 integral membrane protein STOM 6 P84077 ADP-ribosylation factor1; ADP-ribosylation factor 3; ADP-ribosylation factor 5; ADP- ARF1;ARF3; 6 ribosylation factor 4 ARF5; ARF4 P61981 14-3-3 protein gamma;14-3-3 protein gamma, N-terminally processed YWHAG 5 P09211 GlutathioneS-transferase P GSTP1 5 P13489 Ribonuclease inhibitor RNH1 5 Q96PU5 E3ubiquitin-protein ligase NEDD4-like NEDD4L 5 Q5SRN7 HLA class Ihistocompatibility antigen, A; HLA class I histocompatibility antigen,B; HLA HLA-A; 5 class I histocompatibility antigen, Cw HLA-C; HLA-BP02042 Hemoglobin subunit delta HBD 5 A0A087WU29 Glycophorin-A GYPA 5P00390 Glutathione reductase, mitochondrial GSR 5 Q9UBV8 Peflin PEF1 5Q8WVM8 Sec1 family domain-containing protein 1 SCFD1 5 F6TLX2 Glyoxalasedomain-containing protein 4 GLOD4 5 H3BQF1 Adeninephosphoribosyltransferase APRT 5 Q07960 Rho GTPase-activating protein 1ARHGAP1 5 F6XSS0 Blood group Rh(CE) polypeptide; Blood group Rh(D)polypeptide RHCE; RHD 5 A0A087WY55 Vacuolar protein sorting-associatedprotein VTA1 homolog VTA1 5 G5E9W8 Glycogenin-1 GYG1 5 P07451 Carbonicanhydrase 3 CA3 5 Q08722 Leukocyte surface antigen CD47 CD47 5 X6RA14S-formylglutathione hydrolase ESD 5 Q8IUI8 Cytokine receptor-like factor3 CRLF3 5 Q5VW32 BRO1 domain-containing protein BROX BROX 5 P61026Ras-related protein Rab-10 RAB10 5 A0A087WWY3 Filamin-A FLNA 5 H0YGX7Rho GDP-dissociation inhibitor 2 ARHGDIB 5 Q92783 Signal transducingadapter molecule 1 STAM 5 Q7Z6Z7 E3 ubiquitin-protein ligase HUWE1 HUWE15 H0YHC3 Nucleosome assembly protein 1-like 1 NAP1L1 5 Q5QPM7 Proteasomeinhibitor PI31 subunit PSMF1 5 P09104 Gamma-enolase; Enolase ENO2 5Q5T2B5 Cullin-2 CUL2 5 Q8WW22 DnaJ homolog subfamily A member 4 DNAJA4 5P61201 COP9 signalosome complex subunit 2 COPS2 5 X6R433Protein-tyrosine-phosphatase; Receptor-type tyrosine-protein phosphataseC PTPRC 5 P63000 Ras-related C3 botulinum toxin substrate 1 RAC1 5F5GXM3 IST1 homolog IST1 5 H3BLU7 Aflatoxin B1 aldehyde reductase member2 AKR7A2 5 P25325 3-mercaptopyruvatesulfurtransferase; SulfurtransferaseMPST 5 A0A087X0K1 Calcium-binding protein 39 CAB39 5 P23381Tryptophan--tRNA ligase, cytoplasmic; T1-TrpRS; T2-TrpRS WARS 5 P01116GTPase KRas; GTPase KRas, N-terminally processed KRAS 5 P30040Endoplasmic reticulum resident protein 29 ERP29 5 P05198 Eukaryotictranslation initiation factor 2 subunit 1 EIF2S1 5 Q6UX06 Olfactomedin-4OLFM4 5 Q96KP4 Cytosolic non-specific dipeptidase CNDP2 5 Q04760Lactoylglutathione lyase GLO1 5 J3QS39 Ubiquitin-60S ribosomal proteinL40; Ubiquitin; 60S ribosomal protein L40; Ubiquitin-40S UBB; RPS27A; 5ribosomal protein S27a; Ubiquitin; 40S ribosomal protein S27a;Polyubiquitin- UBC; UBA52; B; Ubiquitin; Polyubiquitin-C; UbiquitinUBBP4 P06702 Protein S100-A9 S100A9 5 P15531 Nucleoside diphosphatekinase A; Nucleoside diphosphate kinase; Nucleoside diphosphate NME1;NME2; 5 kinase B NME1-NME2 E7EV99 Alpha-adducin ADD1 5 P51149Ras-related protein Rab-7a RAB7A 4 K7N7A8 Aquaporin-1 AQP1 4 P61020Ras-related protein Rab-5B RAB5B 4 X6R4N5 Erythroid membrane-associatedprotein ERMAP 4 E5RJR5 S-phase kinase-associated protein 1 SKP1 4 Q9Y315Deoxyribose-phosphate aldolase DERA 4 X6R8F3 Neutrophilgelatinase-associated lipocalin LCN2 4 O75396 Vesicle-traffickingprotein SEC22b SEC22B 4 Q15102 Platelet-activating factoracetylhydrolase IB subunit gamma PAFAH1B3 4 P51665 26S proteasomenon-ATPase regulatory subunit 7 PSMD7 4 Q96FZ7 Charged multivesicularbody protein 6 CHMP6 4 Q9NRQ2 Phospholipid scramblase 4 PLSCR4 4 F8VWS060S acidic ribosomal protein P0; 60S acidic ribosomal protein P0-likeRPLP0; 4 RPLP0P6 O14964 Hepatocyte growth factor-regulated tyrosinekinase substrate HGS 4 A0A0A0MTJ9 Neutral cholesterol ester hydrolase 1NCEH1 4 A0A087WY82 Junctional adhesion molecule A F11R 4 J3QSB7 Purinenucleoside phosphorylase; S-methyl-5-thioadenosine phosphorylase MTAP 4Q5WQ6 Ubiquitin thioesterase OTU1 YOD1 4 P36543 V-type proton ATPasesubunit E 1 ATP6V1E1 4 Q9BS40 Latexin LXN 4 P47755 F-actin-cappingprotein subunit alpha-2 CAPZA2 4 Q9GZT8 NIF3-like protein 1 NIF3L1 4H6UYS7 Alpha-synuclein SNCA 4 P60953 Cell division control protein 42homolog CDC42 4 Q14773 Intercellular adhesion molecule 4 ICAM4 4 H0Y6T7Nicastrin NCSTN 4 P69891 Hemoglobin subunit gamma-1 HBG1 4 P14625Endoplasmin HSP90B1 4 Q96GD0 Pyridoxal phosphate phosphatase PDXP 4P08311 Cathepsin G CTSG 4 Q9H9Q2 COP9 signalosome complex subunit 7bCOPS7B 4 P09417 Dihydropteridine reductase QDPR 4 F8WE6 Peptidyl-prolylcis-trans isomerase; Peptidyl-prolyl cis-trans isomerase A PPIA 4 O15173Membrane-associated progesterone receptor component 2 PGRMC2 4 P28072Proteasome subunit beta type-6; Proteasome subunit beta type PSMB6 4H3BSW0 Leucine-rich repeat-containing protein 57 LRRC57 4 Q9UBW8 COP9signalosome complex subunit 7a COPS7A 4 O00560 Syntenin-1 SDCBP 4 F5H157Ras-related protein Rab-35 RAB35 4 Q9H479 Fructosamine-3-kinase FN3K 4Q04917 14-3-3 protein eta YWHAH 4 C9JJ47 AP-2 complex subunit mu AP2M1 4C9JIG9 Serine/threonine-protein kinase OSR1 OXSR1 4 Q13336 Ureatransporter 1 SLC14A1 4 P46926 Glucosamine-6-phosphate isomerase 1;Glucosamine-6-phosphate GNPDA1; 4 isomerase; Glucosamine-6-phosphateisomerase 2 GNPDA2 O43633 Charged multivesicular body protein 2a CHMP2A4 F8VVB9 Tubulin alpha-1A chain; Tubulin alpha-1C chain; Tubulinalpha-1B chain; Tubulin alpha-3C/D TUBA1B; TUBA1C; 4 chain; Tubulinalpha-3E chain TUBA1A; TUBA3C; TUBA3E A6PVN5 Serine/threonine-proteinphosphatase 2A activator PPP2R4 4 B8ZZB8 CB1 cannabinoidreceptor-interacting protein 1 CNRIP1 4 Q9P2R3 Rabankyrin-5 ANKFY1 4Q86YS7 C2 domain-containing protein 5 C2CD5 4 R4GMR5 26S proteasomenon-ATPase regulatory subunit 8 PSMD8 4 E7EM64 COP9 signalosome complexsubunit 6 COPS6 4 J3KNI6 lntegrin beta; Integrin beta-2 ITGB2 4 H0Y5R6Uroporphyrinogen decarboxylase UROD 4 M0R165 Epidermal growth factorreceptor substrate 15-like 1 EPS15L1 4 O00487 26S proteasome non-ATPaseregulatory subunit 14 PSMD14 4 Q13630 GDP-L-fucose synthase TSTA3 4P55060 Exportin-2 CSE1L 4 P20020 Plasma membrane calcium-transportingATPase 1; Calcium-transporting ATPase ATP2B1 4 Q9NYU2 UDP-glucose:glycoprotein glucosyltransferase 1 UGGT1 4 H3BND8 Ubiquitincarboxyl-terminal hydrolase; Ubiquitin carboxyl-terminal hydrolase 7USP7 4 Q9GZP4 PITH domain-containing protein 1 PITHD1 4 D6RD66 WDrepeat-containing protein 1 WDR1 4 P48729 Casein kinase I isoform alphaCSNK1A1 4 P25685 DnaJ homolog subfamily B member 1 DNAJB1 4 P14550Alcohol dehydrogenase [NADP(+)] AKR1A1 4 Q6PCE3 Glucose 1,6-bisphosphatesynthase PGM2L1 4 Q9UPN7 Serine/threonine-protein phosphatase 6regulatory subunit 1 PPP6R1 4 P62805 Histone H4 HIST1H4A 4 P62937Peptidyl-prolyl cis-trans isomerase A PPIA 4 P30046 D-dopachromedecarboxylase; D-dopachrome decarboxylase-like protein DDT; DDTL 4Q08495 Dematin DMTN 4 K7EIJ0 WW domain-binding protein 2 WBP2 4 E5RHP7Carbonic anhydrase 1 CA1 4 P09105 Hemoglobin subunit theta-1 HBQ1 4E7ESC6 Exportin-7 XPO7 4 P61225 Ras-related protein Rap-2b; Ras-relatedprotein Rap-2c; Ras-related protein Rap-2a RAP2B; RAP2A; 4 RAP2C O15400Syntaxin-7 STX7 3 Q5VZR0 Golgi-associated plant pathogenesis-relatedprotein 1 GLIPR2 3 P40199 Carcinoembryonic antigen-related cell adhesionmolecule 6 CEACAM6 3 P27348 14-3-3 protein theta YWHAG 3 P62820Ras-related protein Rab-1A RAB1A 3 C9JEN3 Protein lifeguard 3 TMBIM1 3Q9UDX3 SEC14-like protein 4 SEC14L4 3 Q9Y570 Protein phosphatasemethylesterase 1 PPME1 3 Q96GG9 DCN1-like protein 1; DCN1-like proteinDCUN1D1 3 B0YJC4 Vimentin VIM 3 Q86VN1 Vacuolarprotein-sorting-associated protein 36 VPS36 3 F8WFB9 Endophilin-B2SH3GLB2 3 A0A087WVQ9 Elongation factor 1-alpha 1; Putative elongationfactor 1-alpha-like 3 EEF1A1; 3 EEF1A1P5 Q53TN4 Cytochrome b reductase 1CYBRD1 3 P10809 60 kDa heat shock protein, mitochondrial HSPD1 3 F5H7X126S proteasome non-ATPase regulatory subunit 9 PSMD9 3 E9PNW4 CD59glycoprotein CD59 3 E9PS74 Solute carrier family 43 member 3 SLC43A3 3P53985 Monocarboxylate transporter 1 SLC16A1 3 Q8WWI5 Cholinetransporter-like protein 1 SLC44A1 3 E9PIR7 Thioredoxin reductase 1,cytoplasmic TXNRD1 3 Q5TD07 Ribosyldihydronicotinamide dehydrogenase[quinone] NQO2 3 Q9UL25 Ras-related protein Rab-21 RAB21 3 P27824Calnexin CANX 3 U3KPS2 Myeloblastin PRTN3 3 Q9UKU0Long-chain-fatty-acid--CoA ligase 6 ACSL6 3 P24666 Low molecular weightphosphotyrosine protein phosphatase ACP1 3 H7C2G2 NAD(P)(+)--arginineADP-ribosyltransferase; Ecto-ADP-ribosyltransferase 4 ART4 3 I3L1K6Myosin light chain 4 MYL4 3 Q9UK41 Vacuolar protein sorting-associatedprotein 28 homolog VPS28 3 Q9NUQ9 Protein FAM49B FAM49B 3 Q9UBQ7Glyoxylate reductase/hydroxypyruvate reductase GRHPR 3 B8ZZG1 MAGUK p55subfamily member 6 MPP6 3 Q10567 AP-1 complex subunit beta-1 AP1B1 3O75387 Large neutral amino acids transporter small subunit 3 SLC43A1 3Q9BTU6 Phosphatidylinositol 4-kinase type 2-alpha PI4K2A 3 J3KS22L-xylulose reductase DCXR 3 O15498 Synaptobrevin homolog YKT6 YKT6 3Q08211 ATP-dependent RNA helicase A DHX9 3 P50416 CarnitineO-palmitoyltransferase 1, liver isoform CPT1A 3 P08237 ATP-dependent6-phosphofructokinase, muscle type PFKM 3 P14735 Insulin-degradingenzyme IDE 3 Q9H0R3 Transmembrane protein 222 TMEM222 3 Q14166Tubulin--tyrosine ligase-like protein 12 TTLL12 3 Q14558 Phosphoribosylpyrophosphate synthase-associated protein 1 PRPSAP1 3 A0A087WTB8Ubiquitin carboxyl-terminal hydrolase; Ubiquitin carboxyl-terminalhydrolase isozyme L3 UCHL3 3 Q9NPQ8 Synembryn-A RIC8A 3 F6WQW2Ran-specific GTPase-activating protein RANBP1 3 Q15691Microtubule-associated protein RP/EB family member 1 MAPRE1 3 A0A0A0MR50Cullin-4A CUL4A 3 Q96IU4 Alpha/beta hydrolase domain-containing protein14B ABHD14B 3 Q9P0L0 Vesicle-associated membrane protein-associatedprotein A VAPA 3 E9PRY8 Elongation factor 1-delta EEF1D 3 Q16543 Hsp90co-chaperone Cdc37 CDC37 3 P06702 Protein S100-A9 S100A9 3 P13639Elongation factor 2 EEF2 3 E9PJC7 Tetraspanin; CD82 antigen CD82 3Q01432 AMP deaminase 3 AMPD3 3 B1AUU8 Epidermal growth factor receptorsubstrate 15 EPS15 3 P54709 Sodium/potassium-transporting ATPase subunitbeta-3 ATP1B3 3 P54727 UV excision repair protein RAD23 homolog B RAD23B3 P36507 Dual specificity mitogen-activated protein kinase kinase 2MAP2K2 3 Q9Y2V2 Calcium-regulated heat stable protein 1 CARHSP1 3 P31146Coronin-1A; Coronin CORO1A 3 Q8IY17 Neuropathy target esterase PNPLA6 3E7EQR4 Ezrin EZR 3 F8WDS9 LanC-like protein 1 LANCL1 3 X6RJP6Transgelin-2 TAGLN2 3 A0A0J9YXM6 WD repeat-containing protein 81 WDR81 3P62140 Serine/threonine-protein phosphatase PP1-beta catalytic subunit;Serine/threonine-protein PPP1CB 3 phosphatase H0YLJ3 Mortality factor4-like protein 1 MORF4L1 3 E7EPV7 Alpha-synuclein SNCA 3 K7EK07 HistoneH3 H3F3B 3 P05109 Protein S100-A8; Protein S100-A8, N-terminallyprocessed S100A8 3 I3L0A0 Ubiquitin-conjugating enzyme E2 variant 1;Ubiquitin-conjugating enzyme E2 variant 2 TMEM189- 3 UBE2V1; UBE2V1;UBE2V2 Q6B0K9 Hemoglobin subunit mu HBM 3 O75368 SH3 domain-bindingglutamic acid-rich-like protein SH3BGRL 3 A0A0J9YXB3 Ras-related proteinRap-1b; Ras-related protein Rap-1A; Ras-related protein Rap-1b-likeRAP1B; RAP1A 3 protein P60953 Cell division control protein 42 homologCDC42 3 A0A087WTI1 Ras-related protein Rab-1B; Ras-related proteinRab-1A RAB1B; RAB1A 3 B5MDF5 GTP-binding nuclear protein Ran RAN 3P61088 Ubiquitin-conjugating enzyme E2 N UBE2N 3 Q00013 55 kDaerythrocyte membrane protein MPP1 3 B1AKQ8 Guanine nucleotide-bindingprotein G(I)/G(S)/G(T) subunit beta-1; Guanine nucleotide- GNB1; GNB2; 3binding protein G(I)/G(S)/G(T) subunit beta-3; Guaninenucleotide-binding protein GNB3 G(I)/G(S)/G(T) subunit beta-2 P04406Glyceraldehyde-3-phosphate dehydrogenase GAPDH 3 Q9UJC5 SH3domain-binding glutamic acid-rich-like protein 2 SH3BGRL2 3 P04921Glycophorin-C GYPC 2 Q9NP59 Solute carrier family 40 member 1 SLC40A1 2K7EKH5 Fructose-bisphosphate aldolase; Fructose-bisphosphate aldolase CALDOC 2 P11233 Ras-related protein Ral-A RALA 2 F8WBR5 Calmodulin CALM2;2 CALM3; CALM1 P00441 Superoxide dismutase [Cu—Zn] SOD1 2 H0YDI1Lymphocyte function-associated antigen 3 CD58 2 H0YNE9 Ras-relatedprotein Rab-8B RAB8B 2 J3KN67 TPM3 2 P20160 Azurocidin AZU1 2 A0A087WZZ4Ammonium transporter Rh type A RHAG 2 F5GXS0 Complement C4-A; ComplementC4-B C4B; C4A 2 Q8ND76 Cyclin-Y CCNY 2 Q14739 Lamin-B receptor LBR 2Q15181 Inorganic pyrophosphatase PPA1 2 J3QS92 Galectin-9 LGALS9 2I3L471 Phosphatidylinositol transfer protein alpha isoform PITPNA 2P01111 GTPase NRas NRAS; KRAS 2 F5H4Q5 Vacuolar proteinsorting-associated protein 37C VPS37C 2 A0A0A0MSW4 Phosphatidylinositoltransfer protein beta isoform PITPNB 2 P67775 Serine/threonine-proteinphosphatase 2A catalytic subunit alpha isoform; Serine/threonine-PPP2CA; 2 protein phosphatase PPP2CB P41091 Eukaryotic translationinitiation factor 2 subunit 3; Putative eukaryotic translationinitiation EIF2S3; 2 factor 2 subunit 3-like protein EIF2S3L A0A087WWS7Syntaxin-binding protein 2 STXBP2 2 F8VQX6 Methyltransferase-likeprotein 7A METTL7A 2 B3KT28 FAS-associated factor 1 FAF1 2 K7EIJ8Katanin p60 ATPase-containing subunit A-like 2 KATNAL2 2 P20339Ras-related protein Rab-5A RAB5A 2 O95456 Proteasome assembly chaperone1 PSMG1 2 K7EK45 Polypyrimidine tract-binding protein 1 PTBP1 2 Q15365Poly(rC)-binding protein 1; Poly(rC)-binding protein 3 PCBP1; 2 PCBP3Q9BSJ8 Extended synaptotagmin-1 ESYT1 2 H3BP35 Diphosphomevalonatedecarboxylase MVD 2 H0Y8C4 Serine/threonine-protein phosphatase 2A 56kDa regulatory subunit delta isoform PPP2R5D 2 K7EP09 Bifunctionalcoenzyme A synthase; Phosphopantetheine adenylyltransferase; Dephospho-COASY 2 CoA kinase F8VNT9 Tetraspanin; CD63 antigen CD63 2 Q8NEV1 Caseinkinase II subunit alpha 3; Casein kinase II subunit alpha CSNK2A3; 2CSNK2A1 F8VTQ5 Heterogeneous nuclear ribonucleoprotein A1 HNRNPA1 2A0A087X2E2 Carcinoembryonic antigen-related cell adhesion molecule 8;Carcinoembryonic antigen- CEACAM8; 2 related cell adhesion molecule 1;Carcinoembryonic antigen-related cell adhesion CEACAM5; molecule 5CEACAM1 H0YAS8 Clusterin; Clusterin beta chain; Clusterin alpha chain;Clusterin CLU 2 E9PP54 Tubulin-specific chaperone cofactor E-likeprotein TBCEL 2 P43034 Platelet-activating factor acetylhydrolase IBsubunit alpha PAFAH1B1 2 Q9NT62 Ubiquitin-like-conjugating enzyme ATG3ATG3 2 K7ERZ3 Perilipin-3 PLIN3 2 Q6DD88 Atlastin-3 ATL3 2 Q15084-3Protein disulfide-isomerase A6 PDIA6 2 K7EQH4 ATP synthase subunitalpha, mitochondrial ATP5A1 2 G3V1U5 Vesicle transport protein GOT1BGOLT1B 2 O00186 Syntaxin-binding protein 3 STXBP3 2 Q5T6W2 Heterogeneousnuclear ribonucleoprotein K HNRNPK 2 H0YEY4 ADP-sugar pyrophosphataseNUDT5 2 P35580 Myosin-10 MYH10 2 F5H081 Solute carrier family 2,facilitated glucose transporter member 4 SLC2A4 2 Q9H1C7 Cysteine-richand transmembrane domain-containing protein 1 CYSTM1 2 K7EJ83Cyclin-dependent kinase 2; Cyclin-dependent kinase 3 CDK3; CDK2 2 C9J352Importin subunit alpha-5; Importin subunit alpha-5, N-terminallyprocessed KPNA1 2 H9KV75 Alpha-actinin-1; Alpha-actinin-4;Alpha-actinin-2; Alpha-actinin-3 ACTN1; ACTN4; 2 ACTN3; ACTN2 A0A0A0MQS1Pyrroline-5-carboxylate reductase; Pyrroline-5-carboxylate reductase 3PYCRL 2 D6RBY0 Rieske domain-containing protein RFESD 2 C9JC71 Lowaffinity immunoglobulin gamma Fc region receptor III-A FCGR3A 2 P51811Membrane transport protein XK XK 2 C9J1G2 DnaJ homolog subfamily Bmember 2 DNAJB2 2 Q9UN37 Vacuolar protein sorting-associated protein 4A;Vacuolar protein sorting-associated protein VPS4A; VPS4B; 2 4B;Fidgetin-like protein 1 FIGNL1 P61160 Actin-related protein 2 ACTR2 2P16930 Fumarylacetoacetase FAH 2 A0A087X0K4 CUB and sushidomain-containing protein 2 CSMD2 2 P10746 Uroporphyrinogen-III synthaseUROS 2 K7EQ02 DAZ-associated protein 1 DAZAP1 2 F5GYN4 Ubiquitinthioesterase OTUB1 OTUB1 2 P10599 Thioredoxin TXN 2 O95197 Reticulon-3RTN3 2 E7ETB3 Aspartyl aminopeptidase DNPEP 2 P14868 Aspartate--tRNAligase, cytoplasmic DARS 2 Q08AM6 Protein VAC14 homolog VAC14 2 Q93034Cullin-5 CUL5 2 Q9BQA1 Methylosome protein 50 WDR77 2 A0A0B4J2G9Ubiquitin-conjugating enzyme E2 L3 UBE2L3 2 Q044461,4-alpha-glucan-branching enzyme GBE1 2 Q96NA2 Rab-interactinglysosomal protein RILP 2 Q92539 Phosphatidate phosphatase LPIN2 LPIN2 2P28482 Mitogen-activated protein kinase 1 MAPK1 2 Q7Z406 Myosin-14;Myosin-11 MYH14; 2 11 Q8IU68 Transmembrane channel-like protein 8 TMC8 2Q96BJ3 Axin interactor, dorsalization-associated protein AIDA 2 P20042Eukaryotic translation initiation factor 2 subunit 2 EIF2S2 2 A0A0A0MQR0Docosahexaenoic acid omega-hydroxylase CYP4F3; Phylloquinoneomega-hydroxylase CYP4F2; CYP4F3; 2 CYP4F2; Cytochrome P450 4F12;Phylloquinone omega-hydroxylase CYP4F11 CYP4F11; CYP4F12 Q9BS26Endoplasmic reticulum resident protein 44 ERP44 2 O00178 GTP-bindingprotein 1 GTPBP1 2 P27797 Calreticulin CALR 2 G3V0E5 Transferrinreceptor protein 1; Transferrin receptor protein 1, serum form TFRC 2P48147 Prolyl endopeptidase PREP 2 H7BZC1 Hippocalcin-like protein 1;Neuron-specific calcium-binding protein hippocalcin; Neurocalcin-HPCAL1; 2 delta NCALD; HPCA F8VPD4 CAD protein; Glutamine-dependentcarbamoyl-phosphate synthase; Aspartate CAD 2 carbamoyltransferase;Dihydroorotase Q9NTJ5 Phosphatidylinositide phosphatase SAC1 SACM1L 2P26447 Protein S100-A4 S100A4 2 Q9UIW2 Plexin-A1 PLXNA1 2 H0YJS0 V-typeproton ATPase subunit D ATP6V1D 2 P25445 Tumor necrosis factor receptorsuperfamily member 6 FAS 2 C9JEU5 Fibrinogen gamma chain FGG 2 F5H562Copper-transporting ATPase 2; WND/140 kDa ATP7B 2 B5MCF3 Protein GUCD1GUCD1 2 Q96TA1 Niban-like protein 1 FAM129B 2 F5GWT4Serine/threonine-protein kinase WNK1 WNK1 2 E9PLT1 Platelet glycoprotein4 CD36 2 Q8TDB8 Solute carrier family 2, facilitated glucose transportermember 14; Solute carrier family 2, SLC2A14; 2 facilitated glucosetransporter member 3 SLC2A3 B4DDD6 Drebrin-like protein DBNL 2 E9PII3Band 4.1-like protein 2 EPB41L2 2 E9PNF5 Glutamine-dependent NAD(+)synthetase NADSYN1 2 K7ELL7 Glucosidase 2 subunit beta PRKCSH 2 Q9BV20Methylthioribose-1 -phosphate isomerase MRI1 2 J3KNB4 Cathelicidinantimicrobial peptide; Antibacterial protein FALL-39; Antibacterialprotein LL-37 CAMP 2 Q5T1Z0 Phospholysine phosphohistidine inorganicpyrophosphate phosphatase LHPP 2 H3BLV0 Complement decay-acceleratingfactor CD55 2 Q9Y3E7 Charged multivesicular body protein 3 CHMP3 2Q5T6H7 Xaa-Pro aminopeptidase 1 XPNPEP1 2 O94779 Contactin-5 CNTN5 2E9PNR2 Ras and Rab interactor 1 RIN1 2 E7ESJ7 Protein FAM114A2 FAM114A22 A0A0A0MS99 Multidrug resistance-associated protein 1 ABCC1 2A0A0G2JM15 Large neutral amino acids transporter small subunit 4 SLC43A22 H0YBF7 Arf-GAP with SH3 domain, ANK repeat and PH domain-containingprotein 1; Arf-GAP with ASAP1; 2 SH3 domain, ANK repeat and PHdomain-containing protein 2 ASAP2 P29992 Guanine nucleotide-bindingprotein subunit alpha-11 GNA11 2 F8WF69 Clathrin light chain A CLTA 2B5MCN0 Atlastin-2 ATL2 2 F8W9F9 Serine/threonine-protein kinase WNK2;Serine/threonine-protein kinase WNK3 WNK2; WNK3 2 A2A3F3 Transientreceptor potential cation channel subfamily M member 3 TRPM3 2 Q5KU26Collectin-12 COLEC12 2 O14523 C2 domain-containing protein 2-like C2CD2L2 Q96DG6 Carboxymethylenebutenolidase homolog CMBL 2 Q5THJ4 Vacuolarprotein sorting-associated protein 13D VPS13D 2 Q02790 Peptidyl-prolylcis-trans isomerase FKBP4; Peptidyl-prolyl cis-trans isomerase FKBP4, N-FKBP4 2 terminally processed A0A0G2JQD2 Glutathione S-transferasetheta-1 GSTT1 2 Q9NV96 Cell cycle control protein 50A; Cell cyclecontrol protein 50B TMEM30A; 2 TMEM30B P62330 ADP-ribosylation factor 6ARF6 2 Q9UPU5 Ubiquitin carboxyl-terminal hydrolase 24 USP24 2 B2R4S9Histone H2B HIST1H2BI 2 P10599 Thioredoxin TXN 2 R4GN98 Protein S100;Protein S100-A6 S100A6 2 H3BS66 Small integral membrane protein 1 SMIM12 Q8NHG7 Small VCP/p97-interacting protein SVIP 2 C9JIG9Serine/threonine-protein kinase OSR1; STE20/SPS1-relatedproline-alanine-rich protein OXSR1; 2 kinase STK39 Q5VZR0Golgi-associated plant pathogenesis-related protein 1 GLIPR2 2 F8WD49Anion exchange protein 3; Anion exchange protein; Anion exchange protein2 SLC4A3; 2 SLC4A2 P61626 Lysozyme C; Lysozyme LYZ 2 Q5T123 SH3domain-binding glutamic acid-rich-like protein 3 SH3BGRL3 2 F5H571Ubiquitin carboxyl-terminal hydrolase 5 USP5 2 Q9H3K6 BolA-like protein2 BOLA2; 2 BOLA2B P05164 Myeloperoxidase; Myeloperoxidase; 89 kDamyeloperoxidase; 84 kDa MPO 2 myeloperoxidase; Myeloperoxidase lightchain; Myeloperoxidase heavy chain P50395 Rab GDP dissociation inhibitorbeta GDI2 2 G3V2U7 Acylphosphatase; Acylphosphatase-1 ACYP1 2 J3KNB4Cathelicidin antimicrobial peptide; Antibacterial protein FALL-39;Antibacterial protein LL-37 CAMP 2 H0Y9X3 Programmed cell death protein6 PDCD6 2 A6NJA2 Ubiquitin carboxyl-terminal hydrolase; Ubiquitincarboxyl-terminal hydrolase 14 USP14 2 P35998 26S protease regulatorysubunit 7 PSMC2 2 P30041 Peroxiredoxin-6 PRDX6 2 P35612 Beta-adducinADD2 2 P00918 Carbonic anhydrase 2 CA2 2 A0A0C4DGH5 Cullin-associatedNEDD8-dissociated protein 1 CAND1 2 C9JZN1 Guanine nucleotide-bindingprotein G(I)/G(S)/G(T) subunit beta-2; Guanine nucleotide- GNB2; 1binding protein subunit beta-4 GNB4 I3L4X8 Integrin beta; Integrinbeta-3 ITGB3 1 H7BZJ3 PDIA3 1 P17066 Heat shock 70 kDa protein 6;Putative heat shock 70 kDa protein 7 HSPA6; 1 HSPA7 J3KT70 Arf-GAP withdual PH domain-containing protein 2 ADAP2; 1 CENTA2 Q9Y6M5 Zinctransporter 1 SLC30A1 1 P54725 UV excision repair protein RAD23 homologA RAD23A 1 F8WD59 40S ribosomal protein SA RPSA; 1 RPSAP58 P06753 1E9PMI6 Methylosome subunit pICln CLNS1A 1 Q9BVK6 Transmembrane emp24domain-containing protein 9 TMED9 1 C9JJV6 Myeloid-associateddifferentiation marker MYADM 1 Q8IXQ3 Uncharacterized protein C9orf40C9orf40 1 Q8NDC0 MAPK-interacting and spindle-stabilizing protein-likeMAPK1IP1L 1 Q99747 Gamma-soluble NSF attachment protein NAPG 1 E7EWE1Ubiquitin-like modifier-activating enzyme 5 UBA5 1 J3KNE3Platelet-activating factor acetylhydrolase IB subunit beta PAFAH1B2 1Q8IUI8 Cytokine receptor-like factor 3 CRLF3 1 Q9BTX7 Alpha-tocopheroltransfer protein-like TTPAL 1 P63261 Actin, cytoplasmic 2; Actin,cytoplasmic 2, N-terminally processed ACTG1 1 P31153S-adenosylmethionine synthase isoform type-2 MAT2A 1 O00560 Syntenin-1SDCBP 1 Q4VB86 Protein 4.1 EPB41 1 Q8WUD1 Ras-related protein Rab-2BRAB2B; 1 DKFZp313C1541 Q9NWV4 UPF0587 protein C1orf123 C1orf123 1 Q9Y2Z0Suppressor of G2 allele of SKP1 homolog SUGT1 1 H0YK48 Tropomyosinalpha-1 chain TPM1 1 H3BS66 Small integral membrane protein 1 SMIM1 1Q5VTS0 Neurensin-1 NRSN1 1 H0Y904 Multidrug resistance-associatedprotein 7 ABCC10 1 P08F94 Fibrocystin PKHD1 1 G3V5X4 Nesprin-2 SYNE2 1P48507 Glutamate--cysteine ligase regulatory subunit GCLM 1 H3BUF4Cyclin-D1-binding protein 1 CCNDBP1 1 F8WB30 Target of Myb protein 1TOM1 1 E5RJI8 CA1 1 S4R3E5 Importin subunit alpha-7 KPNA6 1 E7EVS6 ACTB1 P04206 Ig kappa chain V-III region GOL; Ig kappa chain V-III regionWOL; Ig kappa chain V-III region 1 Ti; Ig kappa chain V-III region SIEQ5SSV3 N(G),N(G)-dimethylarginine dimethylaminohydrolase 2 DDAH2 1H0YG54 Oligoribonuclease, mitochondrial REXO2 1 P08246 Neutrophilelastase ELANE 1 P40926 Malate dehydrogenase, mitochondrial MDH2 1P98172 Ephrin-B1 EFNB1 1 Q96DD7 Protein shisa-4 SHISA4 1 Q71RC9 Smallintegral membrane protein 5 SMIM5 1 P68133 Actin, alpha skeletal muscle;Actin, alpha cardiac muscle 1; Actin, gamma-enteric smooth ACTA1; ACTC1;1 muscle; Actin, aortic smooth muscle ACTG2; ACTA2 H0Y9Q6 Clathrin lightchain B CLTB 1 G3V5P0 KTN1 1 Q9NWX6 Probable tRNA(His)guanylyltransferase THG1L 1 Q15404 Ras suppressor protein 1 RSU1 1B2R4S9 Histone H2B HIST1H2B 1 K7EK06 Phenylalanine--tRNA ligase alphasubunit FARSA 1 H7C3S9 COP9 signalosome complex subunit 8 COPS8 1 J3KRV4Dual specificity mitogen-activated protein kinase kinase 3 MAP2K3 1Q15042 Rab3 GTPase-activating protein catalytic subunit RAB3GAP1 1P01893 Putative HLA class I histocompatibility antigen, alpha chain H;HLA class I histocompatibility HLA-H; HLA-C; 1 antigen, Cw-6 alphachain; HLA class I histocompatibility antigen, B-38 alpha chain; HLAHLA-B class I histocompatibility antigen, B-67 alpha chain; HLA class Ihistocompatibility antigen, B-82 alpha chain; HLA class Ihistocompatibility antigen, B-39 alpha chain; HLA class Ihistocompatibility antigen, Cw-18 alpha chain; HLA class Ihistocompatibility antigen, Cw-7 alpha chain; HLA class Ihistocompatibility antigen, B-42 alpha chain; HLA class Ihistocompatibility antigen, B-14 alpha chain; HLA class Ihistocompatibility antigen, B-8 alpha chain; HLA class Ihistocompatibility antigen, B-7 alpha chain Q9UNW1 Multiple inositolpolyphosphate phosphatase 1 MINPP1 1 Q6B0K9 Hemoglobin subunit mu HBM 1O75915 PRA1 family protein 3 ARL6IP5 1 O95376 E3 ubiquitin-proteinligase ARIH2 ARIH2 1 E9PBW4 Hemoglobin subunit gamma-2 HBG2 1 C9J1X0 WDrepeat-containing protein 91 WDR91 1 Q8NCV1 1 Q9Y4P8 WD repeat domainphosphoinositide-interacting protein 2 WIPI2 1 A0A087WUX6 Proteasomalubiquitin receptor ADRM1 ADRM1 1 Q9NQS7 Inner centromere protein INCENP1 P62805 Histone H4 HIST1H4A 1 Q8TDY2 RB1-inducible coiled-coil protein1 RB1CC1 1 C9JFM5 Syntaxin-4 STX4 1 B0QZ43 Erlin-2; Erlin-1 ERLIN1; 1ERLIN2 C9J8T0 Selenocysteine-specific elongation factor EEFSEC 1 P61224Ras-related protein Rap-1b; Ras-related protein Rap-1b-like proteinRAP1B 1 Q99828 Calcium and integrin-binding protein 1 CIB1 1 Q53GQ0Very-long-chain 3-oxoacyl-CoA reductase HSD17B12 1 H0Y9Q9 ADP-ribosylcyclase/cyclic ADP-ribose hydrolase 2 BST1 1 R4GN98 Protein S100;Protein S100-A6 S100A6 1 Q8TB73 Protein NDNF NDNF 1 Q6P1A2Lysophospholipid acyltransferase 5 LPCAT3 1 F2Z2Y4 Pyridoxal kinase PDXK1 Q9HA65 TBC1 domain family member 17 TBC1D17 1 G3V126 V-type protonATPase subunit H ATP6V1H 1 D6RGE2 Isochorismatase domain-containingprotein 1 ISOC1 1 A0A0B4J222 ADP-ribosylation factor-like protein 15ARL15 1 F8W7W4 Androglobin ADGB 1 Q9GZR7 1 A0A087WVC4 cAMP-dependentprotein kinase catalytic subunit beta PRKACB 1 A8MU39Serine/threonine-protein phosphatase; Serine/threonine-proteinphosphatase 5 PPP5C 1 G3V1N2 HBA2 1 P69892 Hemoglobin subunit gamma-2HBG2 1 F8WEZ0 1-phosphatidylinositol 3-phosphate 5-kinase PIKFYVE 1A0A087WZZ4 Ammonium transporter Rh type A RHAG 1 Q1JUQ3 Peptidyl-prolylcis-trans isomerase; Peptidyl-prolyl cis-trans isomerase FKBP1A FKBP12-1 Exin; FKBP1A F8VWZ5 H2.0-like homeobox protein HLX 1 P62877 E3ubiquitin-protein ligase RBX1; E3 ubiquitin-protein ligase RBX1,N-terminally processed RBX1 1 Q7Z5P9 Mucin-19 MUC19 1 F8VPB3 TPK1 1F8WBF4 Transmembrane protein 50B TMEM50B 1 C9JL85 Myotrophin MTPN 1Q53EQ6 Tigger transposable element-derived protein 5 TIGD5 1 E3W974DNPEP 1 H7BYV1 Interferon-induced transmembrane protein 1;Interferon-induced transmembrane protein IFITM2; IFITM3; 1 2;Interferon-induced transmembrane protein 3 IFITM1 D6RC06 Histidine triadnucleotide-binding protein 1 HINT1 1 E9PNW4 CD59 glycoprotein CD59 1H7C2Z6 Grancalcin GCA 1 Q9H1C7 Cysteine-rich and transmembranedomain-containing protein 1 CYSTM1 1 H0YK07 ATP-dependent Clp proteaseATP-binding subunit clpX-like, mitochondrial CLPX 1 E9PG15 14-3-3protein theta YWHAQ 1 K7ENK9 Vesicle-associated membrane protein 3;Vesicle-associated membrane protein 2 VAMP2; VAMP3 1 F8WDD6 Cationchannel sperm-associated protein subunit gamma CATSPERG 1 P35754Glutaredoxin-1 GLRX 1 Q5THJ4 Vacuolar protein sorting-associated protein13D VPS13D 1 B4E3H6 Transforming acidic coiled-coil-containing protein 1TACC1 1 B0YJC4 Vimentin VIM 1 H7C5R6 AT-rich interactivedomain-containing protein 4B ARID4B 1 P49755 Transmembrane emp24domain-containing protein 10 TMED10 1 E9PMJ3 Ribonuclease inhibitor RNH11 B1AHA9 DNA replication licensing factor MCM5 MCM5 1 P23634 Plasmamembrane calcium-transporting ATPase 4 ATP2B4 1 Q14152 Eukaryotictranslation initiation factor 3 subunit A EIF3A 1 C9IY70 60S ribosomalexport protein NMD3 NMD3 1 A0A087WUS7 Ig delta chain C region IGHD 1P07384 Calpain-1 catalytic subunit CAPN1 1 G3V2C9 Guaninenucleotide-binding protein subunit gamma; Guanine nucleotide-bindingprotein GNG2 1 G(I)/G(S)/G(O) subunit gamma-2 Q9Y2Y8 Proteoglycan 3 PRG31 V9GY70 DNAJB12 1 U5GXS0 MAM and LDL-receptor class A domain-containingprotein 1 MALRD1 1 Q92625 Ankyrin repeat and SAM domain-containingprotein 1A ANKS1A 1

The present description refers to a number of documents, the contents ofwhich are herein incorporated by reference in their entirety.

REFERENCES

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1.-44. (canceled)
 45. An in vitro method for preparing a clinical humanblood sample, the method comprising (a) obtaining a preparation ofisolated erythrocyte-derived extracellular vesicles (EEV) from a bloodsample of a subject having or suspected of having Parkinson's disease;(b) processing the preparation of isolated EEV by separatingextracellular vesicles having a diameter of greater than 100 nm fromextracellular vesicles having a diameter of less than 100 nm, therebyobtaining a processed blood sample enriched for extracellular vesicleshaving a diameter of greater than 100 nm; and (c) quantifying theexpression levels of one or more protein biomarkers in the processedblood sample of (b).
 46. The method of claim 45, wherein thequantification in (c) is performed on EEVs having a diameter of between100 nm and 1000 nm.
 47. The method of claim 45, wherein the EEV areCD235a+ extracellular vesicles.
 48. The method of claim 45, wherein theEEV are TSG101+, Rabs+, CD9+, CD63+, CD81+, or any combination thereof.49. The method of claim 45, wherein said blood sample is platelet-freeplasma.
 50. The method of claim 45, further comprising, prior to (a),inducing calcium-dependent production of EEV from activated erythrocytesin the blood sample of the subject.
 51. The method of claim 45, whereinthe preparation of isolated EEV is obtained by separating the EEV byflow cytometry, differential centrifugation, nanomembraneultrafiltration, immunoabsorbent capture, size-exclusion chromatography,ultracentrifugation, magnetic activated cell sorting (MACS),nanoparticle tracking analysis, light scattering, electrophoretic lightscattering, dynamic light scattering, electron microscopy, or anycombination thereof.
 52. The method of claim 45, further comprisingremoving hemoglobin from the preparation of isolated EEV prior to thequantification in (c).
 53. The method of claim 45, wherein thequantification in (c) comprises contacting the one or more proteinbiomarkers with antibodies directed against each of the proteinbiomarkers.
 54. The method of claim 45, wherein the quantification in(c) comprises mass spectrometry.
 55. The method of claim 45, wherein thequantification in (c) comprises nano liquid chromatography tandem massspectrometry (nanoLC MS/MS).
 56. An in vitro method for preparing aclinical human blood sample, the method comprising (a) receiving apreparation of isolated erythrocyte-derived extracellular vesicles (EEV)enriched for EEV having a diameter of greater than 100 nm, from a bloodsample of a subject having or suspected of having Parkinson's disease;and (b) quantifying the expression levels of one or more proteinbiomarkers, in the preparation of isolated EEV enriched for EEV having adiameter of greater than 100 nm.